Generation of neural stem cells from human trophoblast stem cells

ABSTRACT

Provided herein are isolated neural stem cells. Also provided are methods for treatment of neurodegenerative diseases using suitable preparations comprising the isolated neural stem cells.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/413,892, filed Nov. 15, 2010, and U.S. Provisional Application No.61/434,790, filed Jan. 20, 2011, which applications are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 6, 2012, isnamed 38219726.txt and is 12, 129 bytes in size.

BACKGROUND OF THE INVENTION

The human trophoblast stem (hTS) cell is capable of indefiniteproliferation in vitro in an undifferentiated state. The hTS cellmaintains the potential multilineage differentiation capabilities. ThehTS cell preparation can be induced to differentiate into cells of thetrophoblast lineage in vitro or in vivo. Further, hTS cells can beinduced to differentiate into neurons, such as dopaminergic neurons. ThehTS cells can be used to treat a dysfunction or loss of the dopaminergicneurons in the nigrostriatal pathway, such as neurodegenerativedisorders in humans.

SUMMARY OF THE INVENTION

Neurodegenerative disorders have profound socio-economic effects in thehuman population. Current drugs provide only limited benefit byalleviating certain symptoms of neurodegenerative disorders such asParkinson's disease, Alzheimer's disease, Huntington's disease or thelike. Parkinson's disease (PD) is caused by the dysfunction or loss ofthe dopaminergic neurons in the nigrostriatal pathway, and is a commonneurodegenerative disorder in humans. Provided herein are isolatedneural stem cells for alternative cell-based therapy inneurodegenerative disorders, including, Parkinson's disease,Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis(ALS), multiple system atrophy, Lewy body dementia, peripheral sensoryneuropathies or spinal cord injuries in mammals.

Provided herein, in one aspect, are isolated neural stem cells, whereinsaid isolated neural stem cells are derived from trophoblast tissue. Insome embodiments, the trophoblast tissue is human trophoblast tissue.

In one embodiment, an isolated neural stem cell described hereinexpresses transcripts for one or more of caudal type homeobox 2 (Cdx2),Nanog homeobox, nestin, octamer-binding transcription factor 4(Oct-4),neurofilament, neurogenin-3 (Ngn3), neomycin-deleted gene (Neo-D),microtubule-associated protein-2 (MAP-2), CD133, retinoic acid receptorbeta (RARβ), retinoid X receptor alpha (RXRα), retinoid X receptor beta(RXRβ), cellular retinoic acid binding protein 2 (CRABP-2), cellularretinol binding protein 1 (CRBP-1), retinaldehyde dehydrogenase 2(RALDH-2) or retinaldehyde dehydrogenase 3 (RALDH-3).

In one embodiment, the isolated neural stem cell is a human neural stemcell. In one embodiment, the cell has a normal karyotype. In anotherembodiment, the isolated neural stem cell has one or moreimmune-privileged characteristics. In another embodiment, the one ormore immune-privileged characteristics comprise absence of CD33expression and/or CD133 expression.

Further provided herein are methods of differentiating the isolatedneural stem cells into neurons, the method comprising: administeringsaid isolated neural stem cell into the brain of a mammal, wherein saidisolated neural stem cell differentiates into a neuron. In anotherembodiment, the neuron is a dopaminergic neuron, glutaminergic neuron,serotonergic neuron, or GABAergic (gamma aminobutyric acid) neuron.

In one embodiment, the administered (e.g., transplanted) isolated neuralstem cells are pre-induced with an induction drug prior to saidadministering. In another embodiment, the isolated neural stem cells arenot pre-induced with an induction drug prior to said administering.

In one embodiment, the brain of said mammal is damaged or has sufferedneuronal loss, prior to said administering. In another embodiment, saiddamage is to a dopaminergic neuron, glutaminergic neuron, serotonergicneuron, or GABAergic (gamma aminobutyric acid) neuron. In anotherembodiment, said neuronal loss is to a dopaminergic neuron.

In one embodiment, said cell is transfected with an expression vector.

In another embodiment, the isolated neural stem cells, after beingadministered into the brain of said subject, migrate to substantia nigrapars compacta (SNC) region of the brain of the subject. In anotherembodiment, said administration improves sensorimotor function in saidmammal. In another embodiment, said administration causes a reduction insaid mammal's rigidity, akinesia or balance impairment.

Provided herein are methods of differentiating isolated neural stemcells into a dopaminergic neuron, the methods comprising: administeringsaid isolated neural stem cells into the brain of a mammal, wherein saidisolated neural stem cells express transcripts for one or more of Cdx2,Nanog, nestin, Oct-4, neurofilament, NgN3, Neo-D, MAP-2, CD133, RARβ,RXRα, RXRβ, CRABP-2, CRBP-1, RALDH-2 or RALDH-3, wherein said brain ofsaid mammal is damaged or has suffered neuronal loss, wherein one ormore of said isolated neural stem cells differentiates into adopaminergic neuron.

Provided herein are methods of differentiating isolated neural stemcells into a dopaminergic neuron, the method comprising: administeringsaid isolated neural stem cells into the brain of a mammal, wherein saidisolated neural stem cell is derived from trophoblast tissue, whereinsaid brain of said mammal is damaged or has suffered neuronal loss,wherein one or more of said isolated neural stem cells differentiatesinto a dopaminergic neuron.

In one embodiment of the methods described above, said administrationimproves sensorimotor function in said mammal. In another embodiment ofthe methods described above, said administration causes a reduction insaid mammal's rigidity, akinesia or balance impairment.

Provided herein are methods of differentiating an isolated humantrophoblastic stem cell into a neural stem cell comprising: modulatingthe activity of a Cdx2, Nanog, nestin, Oct4, neurofilament, Ngn-3,Neo-D, MAP-2, CD133, RARβ, RXRα, RXRβ, CRABP-2, CRBP-1, RALDH-2, orRALDH-3 gene.

Provided herein are methods of differentiating an isolated humantrophoblastic stem cell into a neural stem cell comprising: modulatingthe level of a Cdx2, Nanog, nestin, Oct4, neurofilament, Ngn-3, Neo-D,MAP-2, CD133, RARβ, RXRα, RXRβ, CRABP-2, CRBP-1, RALDH-2, or RALDH-3transcript.

Provided herein are methods of differentiating an isolated humantrophoblastic stem cell into a neural stem cell comprising: modulatingthe level or activity of a Cdx2, Nanog, nestin, Oct4, neurofilament,Ngn-3, Neo-D, MAP-2, CD133 RARβ, RXRα, RXRβ, CRABP-2, CRBP-1, RALDH-2,or RALDH-3 protein.

Provided herein are methods of screening a compound for use in treatmentor prevention of a disease comprising: contacting an isolated humantrophoblastic stem cell with said compound; and detecting a change inthe activity of at least one gene, transcript or protein in said humantrophoblastic stem cell. In one embodiment of the methods describedabove, the activity of at least one gene, transcript or protein in saidhuman trophoblastic stem cell decreases as compared to a comparableisolated human trophoblastic stem cell not contacted with said compound.In another embodiment of the methods described above, the activity of atleast one gene, transcript or protein in said human trophoblastic stemcell increases as compared to a comparable isolated human trophoblasticstem cell not contacted with said compound. In another embodiment of themethods described above, the disease is a neurodegenerative disorder. Inanother embodiment of the methods described above, the disease isParkinson's, Alzheimer's, Schizophrenia, or Amyotrophic lateralsclerosis.

Provided herein are methods of screening a compound for use in treatmentor prevention of a disease comprising: contacting an isolated humantrophoblastic stem cell with said compound; and detecting a change inthe level of at least one transcript or protein in said humantrophoblastic stem cell. In one embodiment of the methods describedabove, the level of at least one transcript or protein in said humantrophoblastic stem cell decreases as compared to an isolated humantrophoblastic stem cell not contacted with said compound. In anotherembodiment of the methods described above, the level of at least onetranscript or protein in said human trophoblastic stem cell increases ascompared to a comparable isolated human trophoblastic stem cell notcontacted with said compound. In another embodiment of the methodsdescribed above, the disease is a neurodegenerative disorder. In anotherembodiment of the methods described above, the disease is Parkinson's,Alzheimer's, Schizophrenia, or Amyotrophic lateral sclerosis.

Provided herein are methods of screening a compound for ability toinduce changes in a cell comprising: contacting an isolated humantrophoblastic stem cell with said compound; and detecting an inductionof differentiation of said human trophoblastic stem cell.

Provided herein are methods of screening a compound for ability toinduce changes in a cell comprising: contacting an isolated neural stemcell with said compound; and detecting an induction of differentiationof said neural stem cell.

Provided herein are methods of screening a compound for use in treatmentor prevention of a disease comprising: contacting an isolated neuralstem cell with said compound; and detecting a change in the activity ofat least one gene, transcript or protein in said neural stem cell. Inone embodiment of the methods described above, the activity of at leastone gene, transcript or protein in said neural stem cell decreases ascompared to a comparable isolated neural stem cell not contacted withsaid compound. In another embodiment of the methods described above, theactivity of at least one gene, transcript or protein in said neural stemcell increases as compared to a comparable isolated neural stem cell notcontacted with said compound. In another embodiment of the methodsdescribed above, the disease is a neurodegenerative disorder. In aparticular embodiment, the disease is Parkinson's, Alzheimer's,Schizophrenia, or Amyotrophic lateral sclerosis.

Provided herein are methods of screening a compound for use in treatmentor prevention of a disease comprising: contacting an isolated neuralstem cell with said compound; and detecting a change in the level of atleast one transcript or protein in said neural stem cell. In oneembodiment of the methods described above, the level of at least onetranscript or protein in said neural stem cell decreases as compared toa comparable isolated neural stem cell not contacted with said compound.In another embodiment of the methods described above, the level of atleast one transcript or protein in said neural stem cell increases ascompared to a comparable isolated neural stem cell not contacted withsaid compound. In another embodiment of the methods described above, thedisease is a neurodegenerative disorder. In another embodiment of themethods described above, the disease is Parkinson's, Alzheimer's,Schizophrenia, or Amyotrophic lateral sclerosis.

One embodiment provided herein describes a method of treating aneurological disorder in a mammal in need thereof comprisingadministering at least one neural stem cell to said mammal, wherein thecell is immune privileged. In another embodiment, said mammal is amouse, rat, pig, dog, monkey, orangutan or ape. In another embodiment,said mammal is a human.

In one embodiment, said mammal in need thereof has one or more symptomsassociated with a neurological disorder. In another embodiment, said oneor more symptoms is selected from the group consisting of rigidity,akinesia, balance impairment, tremor, gait disorder, maldispositionalgait, dementia, excessive swelling (edema), muscle weakness, atrophy inthe lower extremity, movement disorder (chorea), muscle rigidity, aslowing of physical movement (bradykinesia), loss of physical movement(akinesia), forgetfulness, cognitive (intellectual) impairment, loss ofrecognition (agnosia), impaired functions such as decision-making andplanning, hemifacial paralysis, sensory deficits, numbness, tingling,painful paresthesias in the extremities, weakness, cranial nervepalsies, difficulty with speech, eye movements, visual field defects,blindness, hemorrhage, exudates, proximal muscle wasting, dyskinesia,abnormality of tonus in limb muscles, decrease in myotony,incoordination, wrong indication in finger-finger test or finger-nosetest, dysmetria, Holmes-Stewart phenomenon, incomplete or completesystemic paralysis, optic neuritis, multiple vision, ocular motordisturbance such as nystagmus, spastic paralysis, painful tonic seizure,Lhermitte syndrome, ataxia, mogilalia, vesicorectal disturbance,orthostatic hypotension, decrease in motor function, bed wetting, poorverbalization, poor sleep patterns, sleep disturbance, appetitedisturbance, change in weight, psychomotor agitation or retardation,decreased energy, feelings of worthlessness or excessive orinappropriate guilt, difficulty thinking or concentrating, recurrentthoughts of death or suicidal ideation or attempts, fearfulness,anxiety, irritability, brooding or obsessive rumination, excessiveconcern with physical health, panic attacks, and phobias. In anotherembodiment, said neurological disorder is Parkinson's disease,Alzheimer's disease, Huntington's disease, Amyotrophic lateralsclerosis, Friedreich's ataxia, Lewy body disease, spinal muscularatrophy, multiple system atrophy, dementia, schizophrenia, paralysis,multiple sclerosis, spinal cord injuries, brain injuries (e.g., stroke),cranial nerve disorders, peripheral sensory neuropathies, epilepsy,prion disorders, Creutzfeldt-Jakob disease, Alper's disease,cerebellar/spinocerebellar degeneration, Batten disease, corticobasaldegeneration, Bell's palsy, Guillain-Barre Syndrome, Pick's disease, andautism.

Also provided herein, in one embodiment, is a method of treating aneurological disorder in a mammal in need thereof comprisingadministering at least one neural stem cell to said mammal, wherein thecell is immune privileged and derived from trophoblast tissue. Inanother embodiment, the immune privileged cell has low levels of CD33expression. In another embodiment, the immune privileged cell has lowlevels of CD133 expression. In another embodiment, the neuronalprogenitor stem cell does not elicit an immune response. In anotherembodiment, the neuronal progenitor stem cell does not form a tumor. Inanother embodiment, the neural stem cell expresses transcripts for oneor more of Cdx2, Nanog, nestin, Oct-4, neurofilament, NgN3, Neo-D,MAP-2, CD133, RARβ, RXRα, RXRβ, CRABP-2, CRBP-1, RALDH-2 or RALDH-3.

In another embodiment, the method further comprises administering saidone or more neural stem cell into the brain of a mammal, wherein thecell differentiates into a neuron. In another embodiment, saidadministering comprises injecting or implanting. In another embodiment,said neuron is a dopaminergic neuron, glutaminergic neuron, serotonergicneuron, or GABAergic (gamma aminobutyric acid) neuron. In anotherembodiment, said progenitor cell is pre-induced with an induction drugprior to said administering.

Also provided herein in one embodiment is a method of inducing orpromoting a stem cell to differentiate into a cell with neuronalcharacteristics, comprising: (a) contacting the stem cell with aninduction drug; (b) modulating one or more proteins with the inductiondrug in the stem cell, wherein the one or more proteins comprisewingless-type MMTV integration site 2B (Wnt2B), frizzled family receptor6 (Fzd6), disheveled 3 (Dvl3), frequently rearranged in advanced T-celllymphomas 1 (FRAT1), glycogen synthase kinase 3 beta (GSK3β), histonedeacetylase 6 (HDAC6), β-catenin, guanine nucleotide binding proteinsubunit alpha 11 Gq class (Gα_(q/11)), guanine nucleotide bindingprotein beta (Gβ), retinoid X receptor alpha (RXRα), retinoic acidreceptor beta (RARβ), glutamate receptor 1 (GLuR1),phosphoinositide-3-kinase (PI3K), rac-alpha serine/threonine-proteinkinase (AKt1), rac-beta serine/threonine-protein kinase (AKt2),rac-gamma serine/threonine-protein kinase (AKt3), mammalian target ofrapamycin (mTOR), Eukaryotic translation initiation factor 4E-bindingprotein (EIF4EBP), cAMP responsive element binding protein 1 (CREB1),tyrosine hydroxylase (TH), phospholipase C beta (PLC-β),Phosphatidylinositol 4,5-bisphosphate (PIP2),calcium/calmodulin-dependent protein kinase II inhibitor 2 (CaMKII),eukaryotic translation initiation factor 4B (EIF4B), parkin,alpha-synuclein (SNCA), tublin, calcineurin, Collapsin response mediatorprotein 2 (CRMP-2), nuclear factor of activated T-cells (NFAT1),importin, lymphoid enhancer-binding factor 1 (LEFT), Pituitary homeobox2 (Pitx2), myocyte enhancer factor 2A (MEF2A), or E1A binding proteinp300 (EP300); and (c) inducing or promoting the stem cell todifferentiate into a cell with neuronal characteristics.

In one embodiment, the stem cell is a mammalian trophoblast stem cell.In another embodiment, the stem cell is a mammalian embryonic stem cell.In another embodiment, the stem cell is a mammalian induced pluripotentstem cell. In another embodiment, wherein the stem cell is anendodermal, mesodermal, ectodermal or mesenchymal stem cell. In anotherembodiment, the stem cell is from a mouse, rat, human, chimpanzee,gorilla, dog, pig, goat, dolphin, or cow. In another embodiment, thestem cell is from a human. In another embodiment, the stem cell is ahuman trophoblast stem cell. In another embodiment, the cell withneuronal characteristics is a neural stem cell (NSC), dopamine producingcell, dopaminergic neuron, unipolar neuron, bipolar neuron, multipolarneuron, pyramidal cell, Purkinje cell, and anterior horn cell, basketcell, betz cell, Renshaw cell, granule cell, or medium spiny cell.

In one embodiment, the induction drug comprises retinoic acid,nicotinamide or beta-mercaptoethanol, vitamin B12, heparin, putrescine,biotin, or Fe2+, butylated hydroxyanisole, valproic acid, forskolin,5-azacytidine, indomethacin, isobutylmethylxanthine, or insulin. Inanother embodiment, the modulating comprises increasing the activity ofat least one of the one or more proteins. In another embodiment, themodulating comprises increasing the expression of at least one of theone or more proteins. In another embodiment, increasing expressioncomprises increasing the amount of mRNA encoding at least one of the oneor more proteins or increasing the amount of at least one of the one ormore proteins translated from an mRNA. In another embodiment, themodulating comprises decreasing the activity of at least one of the oneor more proteins. In another embodiment, the modulating comprisesdecreasing the expression of at least one of the one or more proteins.In another embodiment, decreasing expression comprises decreasing theamount of mRNA encoding at least one of the one or more proteins ordecreasing the amount of at least one of the one or more proteinstranslated from an mRNA.

Also described herein the method of inducing or promoting a stem cell todifferentiate into a cell with neuronal characteristics, wherein theneuronal characteristics comprises the expression of dopamine, subunitsof the glutamate N-methyl D-aspartate (NMDA) receptor, synapsin I,a-calcium channel marker, growth associated protein 43 (GAP-43),voltage-dependent K+ channel, a voltage-dependent Ca+ channel, or avoltage-dependent Na+ channel.

In one embodiment, the method of inducing or promoting a stem cell todifferentiate into a cell with neuronal characteristics, comprisesmodulating one or more proteins with the induction drug in the stemcell, wherein the one or more proteins is Wnt2B. In another embodiment,Wnt2B is activated. In another embodiment, Wnt2B is inactivated. Inanother embodiment, Wnt2B is activated and then inactivated. In anotherembodiment, Wnt2B is inactivated and then activated. In anotherembodiment, Wnt2B promotes differentiation or proliferation of the stemcell. In another embodiment, Wnt2B promotes or induces dopamineexpression.

In one embodiment, the method of inducing or promoting a stem cell todifferentiate into a cell with neuronal characteristics, comprisesmodulating one or more proteins with the induction drug in the stemcell, wherein the one or more proteins is GSK3β. In another embodiment,GSK3β is activated. In another embodiment, GSK3β is inactivated. Inanother embodiment, GSK3β is activated and then inactivated. In anotherembodiment, GSK3β is inactivated and then activated. In anotherembodiment, GSK3β promotes differentiation or proliferation of the stemcell. In another embodiment, GSK3β modulates microtubule assembly.

In one embodiment, the method of inducing or promoting a stem cell todifferentiate into a cell with neuronal characteristics, comprisesmodulating one or more proteins with the induction drug in the stemcell, wherein the one or more proteins is CREB1. In another embodiment,CREB1 is activated. In another embodiment, CREB1 is inactivated. Inanother embodiment, CREB1 is activated and then inactivated. In anotherembodiment, CREB1 is inactivated and then activated. In anotherembodiment, CREB1 promotes differentiation or proliferation of the stemcell. In another embodiment, CREB1 promotes or induces dopamineexpression.

In one embodiment, the method of inducing or promoting a stem cell todifferentiate into a cell with neuronal characteristics, comprisesmodulating one or more proteins with the induction drug in the stemcell, wherein the one or more proteins is CaMKII. In another embodiment,CaMKII is activated. In another embodiment, CaMKII is inactivated. Inanother embodiment, CaMKII is activated and then inactivated. In anotherembodiment, CaMKII is inactivated and then activated. In anotherembodiment, CaMKII promotes differentiation or proliferation of the stemcell. In another embodiment, CaMKII modulates microtubule assembly.

In one embodiment, the method of inducing or promoting a stem cell todifferentiate into a cell with neuronal characteristics, comprisesmodulating one or more proteins with the induction drug in the stemcell, wherein the one or more proteins is MAPT. In another embodiment,MAPT is activated. In another embodiment, MAPT is inactivated. Inanother embodiment, MAPT is activated and then inactivated. In anotherembodiment, MAPT is inactivated and then activated. In anotherembodiment, MAPT promotes differentiation or proliferation of the stemcell. In another embodiment, MAPT modulates microtubule assembly.

Provided herein in one embodiment is a method of inducing or promoting astem cell to differentiate into a cell with reduced immunogenicity,comprising: (a) contacting the stem cell with an induction drug; (b)modulating one or more proteins with the induction drug in the stemcell, wherein the one or more proteins comprise Wnt2B, Fzd6, Dvl3,FRAT1, GSK3β, HDAC6, β-catenin, Gα_(q/11), Gβ, RXRα, RARβ, GLuR1, PI3K,AKt1, AKt2, AKt3, mTOR, EIF4EBP, CREB1, TH (tyrosine hydroxylase),PLC-β, PIP2, CaMKII, EIF4B, parkin, SNCA, tublin, calcineurin, CRMP-2,NFAT1, importin, LEFT, Pitx2, MEF2A, or EP300; and (c) inducing orpromoting the stem cell to differentiate into a cell with reducedimmunogenicity.

In one embodiment, the stem cell is a mammalian trophoblast stem cell.In another embodiment, the stem cell is a mammalian embryonic stem cell.In another embodiment, the stem cell is a mammalian induced pluripotentstem cell. In another embodiment, wherein the stem cell is anendodermal, mesodermal, ectodermal or mesenchymal stem cell. In anotherembodiment, the stem cell is from a mouse, rat, human, chimpanzee,gorilla, dog, pig, goat, dolphin, or cow. In another embodiment, thestem cell is from a human. In another embodiment, the stem cell is ahuman trophoblast stem cell.

Described herein in one embodiment is the method of inducing orpromoting a stem cell to differentiate into a cell with reducedimmunogenicity, wherein the cell with reduced immunogenicity is a neuralstem cell (NSC), dopamine producing cell, dopaminergic neuron, unipolarneuron, bipolar neuron, multipolar neuron, pyramidal cell, Purkinjecell, and anterior horn cell, basket cell, betz cell, Renshaw cell,granule cell, or medium spiny cell. In another embodiment, the cell withreduced immunogenicity does not induce an immune response or can inhibitan immune response. In another embodiment, the cell with reducedimmunogenicity does not induce an immune response or can inhibit animmune response by a T cell, B cell, macrophage, microglia cell, mastcell, or NK cell.

In one embodiment, the method of inducing or promoting a stem cell todifferentiate into a cell with reduced immunogenicity comprisescontacting the stem cell with an induction drug, wherein the inductiondrug comprises retinoic acid, nicotinamide or beta-mercaptoethanol,vitamin B12, heparin, putrescine, biotin, or Fe2+, butylatedhydroxyanisole, valproic acid, forskolin, 5-azacytidine, indomethacin,isobutylmethylxanthine, or insulin.

In one embodiment, the method of inducing or promoting a stem cell todifferentiate into a cell with reduced immunogenicity comprisesmodulating one or more proteins with the induction drug in the stemcell, wherein modulating comprises increasing the activity of at leastone of the one or more proteins. In another embodiment, said modulatingcomprises increasing the expression of at least one of the one or moreproteins. In another embodiment, increasing expression comprisesincreasing the amount of mRNA encoding at least one of the one or moreproteins or increasing the amount of at least one of the one or moreproteins translated from an mRNA. In another embodiment, said modulatingcomprises decreasing the activity of at least one of the one or moreproteins. In another embodiment, said modulating comprises decreasingthe expression of at least one of the one or more proteins. In anotherembodiment, decreasing expression comprises decreasing the amount ofmRNA encoding at least one of the one or more proteins or decreasing theamount of at least one of the one or more proteins translated from anmRNA.

In one embodiment, the method of inducing or promoting a stem cell todifferentiate into a cell with reduced immunogenicity further comprisesinducing or promoting the stem cell to differentiate into a cell withneuronal characteristics, wherein the neuronal characteristics comprisesthe expression of dopamine, subunits of the glutamate NMDA receptor,synapsin I, a-calcium channel marker, GAP-43, voltage-dependent K+channel, a voltage-dependent Ca+ channel, or a voltage-dependent Na+channel.

In one embodiment, the method of inducing or promoting a stem cell todifferentiate into a cell with reduced immunogenicity comprisesmodulating one or more proteins with the induction drug in the stemcell, wherein the one or more proteins is NFAT. In another embodiment,NFAT is activated. In another embodiment, NFAT is inactivated. Inanother embodiment, NFAT is activated and then inactivated. In anotherembodiment, NFAT is inactivated and then activated. In anotherembodiment, NFAT promotes differentiation or proliferation of the stemcell. In another embodiment, NFAT modulates microtubule assembly.

Also described herein is a method of inducing or promoting a humantrophoblast stem cell to differentiate into a tNSC (trophoblast neuralstem cell) with reduced immunogenicity or that can inhibit an immuneresponse, comprising: (a) contacting the human trophoblast stem cellwith an induction drug; (b) modulating one or more proteins with theinduction drug in the stem cell, wherein the one or more proteinscomprise Wnt2B, Fzd6, Dvl3, FRAT1, GSK3β, HDAC6, β-catenin, Gα_(q/11),Gβ, RXRα, RARβ, GLuR1, PI3K, AKt1, AKt2, AKt3, mTOR, EIF4EBP, CREB1, TH(tyrosine hydroxylase), PLC-β, PIP2, CaMKII, EIF4B, parkin, SNCA,tublin, calcineurin, CRMP-2, NFAT1, importin, LEFT, Pitx2, MEF2A, orEP300; and (c) inducing or promoting the human trophoblast stem cell todifferentiate into a tNSC.

In one embodiment, the method of inducing or promoting a humantrophoblast stem cell to differentiate into a tNSC (trophoblastneurological stem cell) with reduced immunogenicity or that can inhibitan immune response, comprises contacting the human trophoblast stem cellwith an induction drug, wherein the induction drug comprises retinoicacid, nicotinamide or beta-mercaptoethanol, vitamin B12, heparin,putrescine, biotin, or Fe2+, butylated hydroxyanisole, valproic acid,forskolin, 5-azacytidine, indomethacin, isobutylmethylxanthine, orinsulin. In another embodiment, the tNSC does not induce an immuneresponse or can inhibit an immune response by an immune cell. In anotherembodiment, the immune cell is a T cell, B cell, macrophage, microgliacell, mast cell or NK cell.

Also described herein is a method of inhibiting a tumor cell comprising:contacting the tumor cell with a compound; modulating aryl hydrocarbonreceptor (AhR) in the tumor cell; and inhibiting the tumor cell by themodulation. Additionally described herein is a method of decreasingtumor cell growth comprising: contacting the tumor cell with atherapeutic agent; modulating AhR in the tumor cell; and decreasinggrowth in the tumor cell by the modulation. In one embodiment modulatingAhR comprises inhibiting AhR protein activity in said cell. In anotherembodiment modulating AhR comprises inhibiting AhR gene expression insaid cell. In another embodiment the tumor cell is killed. In anotherembodiment the tumor is a lung, breast, colon, brain, bone, liver,prostate, stomach, esophageal, skin or leukemia tumor. In anotherembodiment tumor is a solid or liquid tumor. In another embodiment AhRis modulated with an AhR agonist. In another embodiment AhR is modulatedwith an AhR antagonist. In another embodiment AhR is modulated with acompound that has anti-estrogenic activity. In another embodiment AhR ismodulated with a compound that has anti-androgenic activity. In anotherembodiment the tumor cell is in a mammal. In another embodiment thetumor cell is in a human.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF FIGURES

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages described herein will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows characteristics of pluripotence and renewal in hTS cells.(1 a) hTS cells express specific genes of both inner cell mass (ICM) andtrophectoderm measured by RT-PCR analysis. (1 b) Illustrates expressionand intracellular localization of specific stage embryonic antigen(SSEA)-1, -3, and -4 as visualized by immunocytochemical staining(darkened spots). In hTS cells (upper panels), SSEA-1 is expressedmostly in the cytoplasm (left upper panel), SSEA-3 is expressed in thenucleus (middle upper panel), and SSEA-4 is expressed in both thecytoplasm and membrane (upper right panel). These SSEA-expressed cellswere histologically identical to the ectopic villous cytotrophoblasts(lower panels). (1 c) Unchanged telomere length at 3rd and 7th passagesof hTS cells culture measured by the Terminal Restriction Fragment (TRF)Southern blot analysis (upper and lower panels). (1 d) Venn diagramillustrates the microarray analysis of gene expression in hTS (859genes) and trophoblast associated placenta derived mesenchymal stemcells (PDMS cells) (2449 genes). A total of 2,149 and 3,730 genesexpressed in the hTS cells and trophoblast associated PDMS cells (foldchange >2-fold). (1 e) Illustrates results from reverse transcriptionpolymerase chain reaction (RT-PCR) analysis of transcription factorexpression in response to different concentrations of leukemiainhibitory factor (LIF) (i.e., 500, 250, 125 U/ml; U: units/ml, Actin:β-actin as the control sample). Withdrawal of LIF suppresses Oct4 andSox2, but overexpresses Nanog and Cdx2 in hTS cells. (1 f) Flowcytometric analysis of LIF (125 U/ml) promoted expressions of Nanog,Cdx2, Sox2, and Oct4 in hTS cells (left panel). Histogram shows anegative dose-dependent manner in Nanog and Cdx2 (left panels) and apositive dose-dependent manner in Oct4 and Sox2 (right panel). (1 g) Adiagram of the physiological distribution of LIF levels in the differentsegments of fallopian tubes in women, specifically the physiologicalreduction of LIF levels from ampulla toward isthmus in the fallopiantubes. The relative ratio of Oct4, Nanog and Sox2 to Cdx2 each show adose-dependency in three different segments of the fallopian tube. (1 h)Effect of different siRNAs to specific transcriptors Nanog and Cdx2 wasanalyzed by RT-PCR (left) and flow cytometric analyses (right) in hTScells, illustrating a reciprocal relationship between Nanog and Cdx2 inthe maintenance of pluripotency of hTS cells. Data indicated mean±SD for3 assays. (1 i) Histogram of gene intensity shows a homogeneous patternin hTS cells, while PDMS cells show a biphasic pattern.

FIG. 2 illustrates retinoic acid (RA) induced hTS cell differentiationinto a variety of phenotypical neural stem cells. (2 a) Distribution ofvarious neural progenitor subtypes, including glial restrictedprecursors (GRP), neuronal restricted precursors (NRP), multipotentneural stem (MNS) cells, astrocytes (AST), and undefined trophoblastgiant cells (TGC). The frequency of the hTS cell-derived neuralprogenitor subtypes distributed in consistent ratios during RA inductionwith time, (e.g. 1, 3, 5 and 7 days), shown from the first to the fourthrow, respectively. n: indicating total cell number counted. (2 b) RT-PCRanalysis of hTS cell expression of neural stem cell-related genes beforeand after 1-day RA (10 μM) induction, including nestin, Oct4,neurofilament, Ngn3, Neo-D, MAP-2 and CD133, generated from RA (10 μM)induced hTS cells. (2 c) Both 3- and 5-day RA-induced hTS cellsexpressed positive immunoreactive neural stem cell genes, includingneurofilament protein, nestin, and GFAP, which sustained a similar ratioin distribution as observed by flow cytometric analysis. (2 d)Immunocytochemical analysis of the (neural stem cells) tNSCs expressedimmunoreactive nestin, tyrosine hydroxylase-2 (TH-2), and serotonin. (2e) Comparative expression of the immune-related genes among hTS cells,tNSCs and (human embryonic stem) hES cells by flow cytometric analysis:HLA-ABC (MHC class I) expressed highly in hTS cells (99.4%) and tNSCsbut lower in hES cells. HLA-DR (MHC class II) did not express in thecells. (2 f) Comparative expression of the immune-related genes amonghTS cells, tNSCs and hES cells by flow cytometric analysis: Nodifference observed in CD14 and CD44 expression among the cells.Proliferative factor CD73 expressed highly in hTS cells and tNSCs, butnegatively expressed in hES cells. (2 g) Comparative expression of theimmune-related genes among hTS cells, tNSCs and hES cells by flowcytometric analysis: transmembrane receptor CD33 is expressed in hTS andhES cells but not in tNSCs. CD45 did not express in the cells. (2 h)Comparative expression of the immune-related genes among hTS cells,tNSCs and hES cells by flow cytometric analysis: no difference inintensities was found among hTS cells, tNSCs and hES in the expressionof mesenchymal stem cell marker CD105, however, less cancer stem cellmarker CD133 (11.8%) was expressed in tNSCs compared to hTS cells(93.6%) and hES cells (98.8%).

FIG. 3 illustrates RA-induced gene expression. (3 a) Illustrates theeffect of RA (10 μM) in the activation c-Src/Stat3/Nanog pathway in thetNSCs. RA induced apparent expression of c-Src, peaking at 15 min andthen sustaining at a lower levels determined by RT-PCR analysis (n=3).(3 b) Shows RA stimulated RXRα, c-Src and RARβ expression at 30 min, 1h, 2 h, and 4 h, respectively, by western blot analysis. RA inductionpromotes both Gαq/11 and Gβ expressions in 30 min, suggesting theinvolvement of G proteins signaling. (3 c) Immunoprecipitation (IP)assays demonstrate RA induced direct binding between RXRα and RARβ;however, this interaction is blocked by c-Src inhibitor PP1 analog,indicating that c-Src is involved in RXRα and RARβ binding to form ascaffolding protein complex. (3 d) IP assay analysis shows that RXRα hasan independent binding interaction with Gαq/11 while RARβ has anindependent binding interaction with Gβ. (3 e) Illustrates a Westernblot analysis of RA induced early production of c-Src, apparentphosphorylation of Stat3 at Tyr705 site and activation of Nanog at 1 hin hTS; β-actin utilized for control sample. (3 f) This rapid productionof c-Src protein then induced phosphorylation of Stat3 at Tyr705 site aswell as overexpression of Nanog by Western blotting assay. The c-Srcinhibitor PP1 analog (4 μM) inhibited the RA-induced phosphorylation ofStat3 at Tyr 705 and expression of Nanog by Western blot analysis. Thisinhibitory action could not be rescued by adding RA. (3 g) Illustratesthe chromatin immunoprecipitation assay (ChIP) assay analysis of RAstimulated binding interaction of Stat3 and Nanog promoter. Input:lysate, C: control.

FIG. 4 illustrates the double immunogold fluorescence transmissionelectron microscopy (IEM) assay results. RA-induced binding interactionbetween the small gold particle-labeled RXRα (6 μm) and the large goldparticle-labeled Gαq/11 (20 μm) at the plasma membrane is shown. Bydynamic confocal immunofluorescence microscopy, immunostained RXRα andGαq/11 appeared primarily in a homogenous feature in either cytoplasm ornucleus (FIG. 4, upper panel). By treatment with RA for 5 min, thecytosolic RXRα intensity increased at the peri-nuclear regions while thenuclear intensity decreased (first column), indicating a cytosolictranslocation after stimulation. The nuclear RXRα intensity becameprominent at 15 min, while the cytosolic intensity decreased. Thesephenomena suggest that an increase of activity in nucleus maintains asteady-status in the cell. An apparent cytosolic translocation wasobserved again in 30 min. The compartmental changes of Gαq/11expression, on the other hand, were similar to that of RXRα (secondcolumn).

FIG. 5 illustrates the analysis of the transplantation of GFP-taggedtNSCs (3×10⁶) into Parkinson's Disease (PD) rats. (5 a) Analysis ofapomorphine induced rotation test; Group a (dark-shaded circles, n=4),which correlates to PD rats that received tNSCs transplantation, showssignificant reduction in contralateral rotation from 3 weeks to 12 weekspostimplantation; Group b (light-shaded circles, n=4), which correlateto PD rats that received 5-day RA-treated hTS cells, shows an initialsignificant improvement at 6 weeks postimplantation but this improvementdecreased gradually through week 12; and Group c (triangles, n=4), whichcorrelates to the untreated PD rats as the control group, shows noimprovement. Statistic analysis by repeated measure ANOVA: p value=0.001and LSD post hoc comparisons after repeated measure ANOVA in between twogroups: p=0.037 (group a vs. c) and p=0.008 (group b vs. c) at 6 weeks;p=0.019 (group a vs. c) at 9 weeks; p=0.005 (group a vs. c) and p=0.018(group a vs. b) at 12 weeks. * indicates p<0.05. (5 b) IllustratesTH-positive immunohistochemical staining in the lesioned striatum ofGroup a at 18 weeks postimplantation (upper panel); immunofluorescencemicroscopic analysis indicates that the immunofluorescent GFP-taggedtNSCs still persisted in the lesioned striatum with a patchy formationat the injection site (lower panel). (5 c) illustrates TH-positiveneurons regenerated in the lesioned substantia nigra compacta (SNC) ofGroup a at 18 weeks postimplantation (upper panel); amplification of theterminal region is shown (lower left panel), Scale bar: 100 μm;immunofluorescence microscopic analysis indicates that theimmunofluorescent GFP-tagged tNSCs persisted in a scattered distribution(lower right panel, arrows indicating GFP-tagged tNSCs). (5 d)Illustrates immunohistochemical staining of Group b at 18 weekspostimplantation: no TH-positive cells were found in the left lesionedstriatum (str, upper panel) or subthalamic nucleus (stn, lower panel).(5 e) Illustrates immunohistochemical staining of Group c at 18 weekspostimplantation: no TH-stained cells were found in the left lesionedstriatum (str, upper panel) or lesioned SNC (lower panel); arrowindicates implanting needle track.

FIG. 6 illustrates the results from transplantation of tNSCs (1.5×10⁶)at one injection site into the lesioned striatum of “aged” PD rats(n=16; body weight, 630-490 gm). Behavioral assessments were analyzedevery 3 weeks postimplantation. Results showed that there was asignificant improvement of behavioral impairments assessed from 3 weeksto 12 weeks postimplanation. Student t test: *p<0.05 as statisticsignificance. **p<0.01 and ***p<0.001. (6 a) Analysis ofapomorphine-induced rotation tests demonstrate aged PD rats thatreceived tNSCs implantation significantly improved the rotation turnsfrom 3 weeks to 12 weeks (group ii, n=8, filled circles) compared to theuntreated “aged” PD rats as the control group (group i, n=8, unfilledcircles). (6 b) Illustrates behavioral assessment results for akinesia(sec). (6 c) Illustrates behavioral assessment results for step length(mm). (6 d) Illustrates behavioral assessment results for stride length(mm). (6 e) Illustrates behavioral assessment results for walking speed(cm/sec). (6 f) Illustrates behavioral assessment results for base ofsupport (mm). (6 g) Illustrates the gaits analyzed for behavioralassessments: A correlates with normal rats, B correlates withhemiparkinsonian rats prior to cell transplantation, and C correlateswith hemiparkinsonian rats after cell transplantation.

FIG. 7 illustrates that hTS cells express components of all threeprimary germ layers, including the ectoderm, the mesoderm and theendoderm after appropriate inductions; left column of each panelcorrelates to gene expression before induction; right column of eachpanel correlates to gene expression after induction.

FIG. 8 illustrates flow cytometric analysis results, indicating that hTScells express mesenchymal stem cell markers (CD90, CD44, CK7, Vimentinand Neurofilament) and are negative for hematopoietic stem cell markers(CD34, CD45, α6-integrin, E-cadherin, and L-selectin).

FIG. 9 shows that upon appropriate induction, hTS cells could bedifferentiated into a variety of specific cell phenotypes.

FIG. 10 illustrates that the histological analysis of transplantation ofhTS cells into the male severe combined immune deficiency (SCID) micesubcutaneously caused only minor chimeric reaction with myxoid-likebizarre cells at 6-8 weeks postimplantation (filled, black arrowsdesignate bizarre cells; unfilled arrows designate muscle fiber; “NT”designates needle track).

FIG. 11. Chromosome analysis showed that hTS cells did not change thepatterns of karyotypes (46, XY). To check the cell lifespan ingenerations, no significant shortening in telomere length was observedbetween 3rd and 7th passage in culture (FIG. 1c ) by Southern blotanalysis.

FIG. 12 illustrates certain media that were used for celldifferentiation.

FIG. 13 illustrates PCR primers (SEQ ID NOS 11-48, respectively, inorder of appearance) that were used for RT-PCR.

FIG. 14 illustrates the analysis of AhR as a signal molecule at theplasma membrane, including the activities of transfected pGFP-C1-AhR atthe plasma membrane by introduction of BBP (1 μM) in Huh-7 cells. (14 a)Images shown are the expressions of relative intensity of GFP-tagged AhRmeasured by TIRF microscopic analysis. The circle and arrow indicate thearea measured over time: before stimulation (first panel), at peak(second panel) and at rest (third panel). The graph (fourth panel) showsthat a peak value was found at around 2 min, with the arrow indicatingtime BBP was added. (14 b) Quantitative RT-PCR analysis of memAhR inresponse to BBP shows a rapid elevation at 5 min peaking at 15 minfollowed by a gradual decline to a lower plateau levels at 2 h. Errorbars indicate standard deviation. *, P<0.05, t-test (n=3). (14 c)Analysis of Western blot assays reveals that BBP promoted AhR elevationat 15 min followed by a slight decrease at 30 min and a re-elevation at60 min. (14 d) Analysis of Western blot assays reveals that BBP inducesthe production of both Gα_(q/11) and Gβ at 30 min. (14 e)Immunoprecipitation (IP) assay indicates the interaction between AhR andG_(αq/11) after BBP stimulation, the letter C representing control. (14f) Knockout of AhR by siRNA demonstrates that BBP suppresses both AhRand Gα_(q/11) expressions measured by Western blot analysis in Huh-7cells, the letter S representing scrambled siRNA as negative control.

FIG. 15 illustrates results of dynamic immunofluorescence imaging. (15a) illustrates immunostaining of untreated control cells; AhR andGα_(q/11) expression observed mainly in the nucleus and weakly in thecytosol in Huh-7 cells; bar scale: 50 μm. (15 b) Cells treated with BBP(1 μM) for 5 and 15 min each reveals a translocation of both AhR andGα_(q/11) from the nucleus to the cytosolic compartment. ImmunostainedGα_(q/11) accumulates specifically at the cell membrane at 15 min. (15c) Cells transfected with AhR siRNA greatly reduces AhR intensity inboth cytosolic and nuclear compartments (upper panel), while transfectedwith scrambled siRNA does not change the immunostaining intensity (lowerpanel). (15 d) BBP rescued intensities of both AhR and Gα_(q/11) incells with pre-transfected AhR siRNA after 15 min.

FIG. 16 illustrates the results of double immunogold transmissionelectron microscopic analysis. (16 a) Immunogold-stained Gα_(q/11)(white arrow) could exist as either single or double or triple in entityat the cell membrane in Huh-7 cells as control. (16 b) At 20 min, BBP(1μM)-treated cell showed an interaction of immunogold-tagged AhRparticles (6 nm in size, black arrow) and immunogold-tagged Gα_(q/11)particles (20 nm in size, white arrow), forming a complex, appearing asdifferent entities: monomeric (not shown), dimeric (not shown), trimeric(left) and polymeric entities (right) at the plasma membrane. (16 c) Atrimeric complex of AhR and Gα_(q/11) appeared at the cell membrane. CM:cell membrane, N: nucleus, and bar scale: 500 nm.

FIG. 17 illustrates the “pull and push” mechanism and biochemicalprocesses. (17 a) illustrates measurements of Gα_(q/11) signalingcascades in response to BBP treatment in Huh-7 cells. Western blotanalysis revealed that BBP (1 μM) triggered production of both Gα_(q/11)and Gβ at 30 min. Activated Gαq_(/11) led to decreases in PIP2, causingincreased IP3R levels. (17 b) illustrates the analysis of theresponsiveness of immunofluorescent Fluo-4-labeled calcium in the Huh-7cells. Shown are the unlabelled cells (left upper panel) andFluo-4-labeled calcium (green, left lower panel). Also shown are thechanges of relative calcium levels after BBP (1 μM) stimulation (arrow)in BSS medium (middle upper panel) and calcium free medium (middle lowerpanel). Cells cultured in the calcium free medium with pre-treated IP3Rinhibitor 2-APB (100 μM, 1 h) (right upper panel) showed a reduction incalcium intensive (right upper panel), which occurred in a dose-responsemanner (y=−0.4x+2.5, R²=0.94) (right lower panel). Error bars indicatethe standard deviation of the mean (n=5). (17 c) Results of Western blotanalysis indicate that the BBP-induced COX-2 expression was inhibited bypretreatment with 2-APB (30 μM, 1 h), the letter C indicating control.(17 d) illustrates results of Western blot analysis, indicating that BBP(1 μM) induced the overproduction of COX-2 via AhR/Ca²⁺/ERK/COX-2pathway. ERK1/2 was phosphorylated at 15 min and dephosphorylated at 30min after BBP treatment. (17 e) illustrates results of Western blotanalysis, indicating that the BBP-induced COX-2 expression was inhibitedby pretreatment with chemical PD98059 (20 μM, 1 h, Calciochem), theletter C indicating control. (17 f) illustrates that ARNT levels weresignificantly inhibited by treatment with BBP (1 μM) measured overnight.Data represent the means±SD, n=3 and *: Student's t-test, p<0.01. (17 g)illustrates a schematic representation of the “pull and push” mechanismunderlying the ligand-induced nongenomic AhR signaling pathway viaGPCRs-G protein signaling.

FIG. 18 illustrates that effect of LIF on Nanog expression. (18 a)illustrates LIF promoted expressions of Nanog. Left panels illustratethat Nanog expression is significantly suppressed in a negativedose-dependent fashion by flow cytometric analysis in hTS cells. Dataindicated mean±SD for three assays. *p<0.01 (Student's t test, n=3).Right panel illustrates relative Nanog expression when hTS cells arepreincubated with RA (10 μM) overnight followed by treating LIF withdifferent levels (i.e., 125, 250 and 500 U/ml each) for 1-day. (18 b)illustrates RA induction (1 day incubation, 10 μM) in hTS cellsstimulated expression of Nanog and Oct4, but not Cdx2 and Sox2 by flowcytometric analysis.

FIG. 19 illustrates the assessment of behavioral improvements in elderlyPD rats. (19 a) illustrates immunohistochemistry of TH+ neurons on aseries of brain sections (30 μm) at 12 weeks postimplantation revealedthat abundant newly regenerated TH-positive neurons appeared in thelesioned nigrostriatal pathway (left portion). In the SNC areas, theTH-positive neurons appeared in a feature with multiple outgrowthsprojecting from the cell body to form neuronal circuitries with the hosttissue. The number of regenerated dopaminergic neurons in one rataccounted for 28.2% of the opposite normal side (n=5). (19 b) The numberof dopaminergic neurons in the lesioned SNC of a rat regenerated to28.2% compared to the normal side.

FIG. 20: (20 a) illustrates the expression of specific genes of both ICMand trophectoderm (TE) by RT-PCR; (20 b) illustrates hTS cells weretransfected with the a DNA mixture of F1B-GFP plasmid construct to yielda success rate of over 95%; (20 c) illustrates time course of RA inducedproduction of eIF4B; (20 d) illustrates activation of c-Src wasinhibited by using eIF4B; (20 e) illustrates IP analysis indicating thatactive c-Src binds directly to Stat3 (signal transducer and activator oftranscription); (20 f) illustrates c-Src siRNA inhibited expression ofStat3; (20 g) illustrates Nanog expression was inhibited by Stst3 siRNA;and (20 h) illustrates a scheme of the RA-induced c-Src/Stat3/Nanogpathway via subcellular c-Src mRNA localization in hTS cells.

FIG. 21 illustrates activation of Gα_(q/11) signaling pathway: (21 a)illustrates expressions of Gα_(q/11) pathway-related components after RAtreatment (10 μM) over time by Western blots; (21 b) illustratesreal-time live cell imaging microscopy (Cell-R system, Olympus, Tokyo)in hTS cells which were cultured in the calcium-free medium andpre-loaded with Fluo4 (1 μM) in BSS buffer 20 min before RA treatment.(a) The RA-induced depletion of intracellular calcium was rescued byadding CaCl₂ (2 mM) with a SOCE pattern. (b) RA-induced intracellularcalcium levels were inhibited by 2-APB (10 min) in a significantdose-dependent manner (R²=0.8984). (c) After depletion of ER calcium,KCl (60 mM) enabled to activate L-type calcium channels. (d)KCl-dependent L-type calcium channels were blocked by inhibitornifedipine (5 μM) after ER calcium depletion. n: total cells counted;(21 c) illustrates that CaMKII directly interacted with CREB1 and eIF4B;(21 d) illustrates eIF4B siRNA inhibited expressions of CaMKII,calcineurin, and eIF4B by Western blots; (21 e) illustrates KN93 (1 μM,2 hr) inhibited eIF4B expression by Western blots; (21 f) illustratesparkin directly interacted with CaMKII and MAPT; (21 g) illustrates SNCAdirectly interacted with MAPT; (21 h) illustrates MAPT interacted withGSK3β and α-tubulin; (21 i) illustrates 2-APB inhibited expressions ofCalcineurin, NFAT1, and MEF2A by Western blots; (21 j) illustratesdirect interaction between Importin and NFAT1; (21 k) illustrates RAstimulated NFAT1 nuclear translocation by fractional assay. Lamin A/C:nuclear marker and α-tubulin: cytopasmic marker; (21 l) illustrates Akt2directly interacted with GSK3β; (21 m) illustrates flow analysis ofGSK3β expression in cells treated with RA for 4 hr (blank column) andfor 24 hr (black column) with different antibodies revealed in dynamicchanges. Data show mean±SD, n=3; (21 n) illustrates flow cytometricanalysis showed that Akt2 siRNA inhibited RA-induced GSK3β expression.

FIG. 22 illustrates formation of transcriptional complex: (22 a)illustrates interaction between β-catenin and LEF1 (upper) and betweenLEF1 and Pitx2; (22 b) illustrates LEF1 transcribed genes Pitx2 gene butnot Pixt3 by RA treatment (4 hr); (22 c) illustrates MEF2A directlyinteracted with NFAT1, MEF2A, Pitx2, SNCA, and EP300 by Western blots;(22 d) illustrates RA induced production of MEF2A, EP300, and Pitx2 overtime by Western blots; (22 e) illustrates NFAT1 siRNA inhibitedexpression of MEF2A by Western blots; (22 f) illustrates CREB1 targetedat the promoter of gene MEF2A; (22 g) illustrates MEF2A transcribedgenes SNCA (upper), TH (middle), and MEF2A itself (lower); (22 h)illustrates MEF2A siRNA inhibited expressions of EP300, Pitx2, and MEF2Aby Western blots; (22 i) illustrates EP300 targeted at promoter of genesHDAC6 (upper) and TH (lower); (22 j) illustrates identification of thevarious molecular activities at time points, 4 hr and 24 hr by Westernblots. Abbreviation, IP: immunoprecipitation assay; ChIP: chromatinimmunoprecipitation assay.

FIG. 23 illustrates the schematic regulatory networks of RA-inducedneurogenesis in hTS cells (upper panel). Two mRNA translationalmachineries: the cap-dependent (left lower) and cap-independent (rightlower). Grey line: the spatiotemporal signaling pathways; black line:the transcriptional pathways; double-headed arrow: molecule in a linkageto other pathway.

FIG. 24 illustrates that RA signaling promotes Wnt2B/Fzd6/β-cateninpathway: (24 a) illustrates flow cytometry analysis indicating that RA(10 μM) induced significantly activations of Wnt2B, Dvl3, and FRAT1 butinhibited GSK3β overnight evidenced by inhibitory action of pretreatedWnt2B siRNA. Data shows mean±SD; n=3; (24 b) illustrates that increasedFzd6 mRNA expression by RA RT-PCR. Data shows mean±SD; n=3, *: p<0.05 byStudent's test; (24 c) illustrates RA induced changes of expression inβ-catenin and HDAC6 over time by Western blots; (24 d) illustrates thatIP assay revealed a physical interaction between HDAC6 and β-catenin byovernight incubation with RA; (24 e) illustrates RA inducednuclear/cytoplasmic translocation of β-catenin by fractionation assayafter overnight incubation. Lamin and α-tubulin serve as nuclear andcytoplasmic markers, respectively; (24 f) illustrates confocalimmunofluorescence microscopy showing dynamic changes of the RA-inducedβ-catenin and HDAC6 indicated nuclear translocation of β-catenin at 30min, which was inhibited by HDAC6 siRNA; (24 g) illustrates thatpunctate β-catenin appeared in the synaptic regions at 5 min of RAtreatment (arrow).

FIG. 25 illustrates confocal immunofluorescence microscopy analysis. Inthe presence of siRNA against HDAC6, nuclear localization of β-cateninwas blocked.

FIG. 26 illustrates molecular events at the cell membrane: (26 a)illustrates RA induced productions of Gα_(q/11), Gβ. RXRα, and RARβ overtime by Western blots. β-actin as control; (26 b) illustrates real-timeconfocal immunofluorescence microscopy analysis, revealing the movementof representative GFP-tagged RXRα from the perinuclear regions towardsthe cell membrane (arrow) after RA stimulation at 0, 4.5, and 13 min. NoRXRα was visible in the nucleus. Normal phase contrast (left upper) andfluorescent image (right upper). Bar indicates 30 μm; (26 c) illustratesa dynamic movement and changes in intensity of the relativelyquantitative GFP-tagged RXRα from the nucleus (N) to the cell membrane(M) in time course. Normal phase contrast and fluorescent imaging showat upper right; (26 d) illustrates that a representative imagingrevealed co-expression of RXRα and Gα_(q/11) at the cell membrane by RAat 5 min; (26 e) illustrates double immunogold labeling of RXRα (6 μm;black arrow) and Gα_(q/11) (20 μm; white arrow) at the cell membraneobserved after RA treatment for 20 min. N: nucleus; (26 f) illustratesRXRα siRNA inhabited the RA-induced interaction of Gα_(q/11) and RXRα(24 hr); (26 g) illustrates RARβ siRNA inhibited the RA-inducedinteraction of Gβ and RARβ as well as interaction of Gβ and PI3K (24hr). IP: immunoprecipitation assay; IgG: negative control; C: positivecontrol; (26 h) illustrates IP assay analysis showing a selective c-Srcinhibitor PP1 analog was able to prevent the formation of RXRα-RARβheterodimer; (26 i) illustrates anchorage of the RA-induced goldparticle-tagged RXRα in the endoplasmic reticulum (ER) observed bydouble immunogold transmission electron microscopy.

FIG. 27 illustrates that RA stimulates canonical Wnt2B pathway byRT-PCR; RA induced expressions of components of Wnt2B signaling pathwayafter overnight treatment (10 μM) in hTS cells, showing in a significantstatistically; Wnt2B siRNA inhibited the RA-induced components of Wnt2Bpathway after overnight treatment.

FIG. 28 illustrates local syntheses of RXRα and RARβ: (28 a) illustratesthat RA (10 μM) induced rapidly transient elevation of both RXRα mRNAand RARβ mRNA at 15 min by RT-PCR. Data show mean±SD, n=3, t test *:p<0.05; (28 b) illustrates that RA induced expressions of PI3K and Aktisoforms over time by Western blots; (28 c) illustrates that PI3Kinhibitor 124005 inhibited the RA-induced Akt isoforms (24 hr) by flowcytometry. Data show mean±SD, n=3; (28 d) illustrates Akt3 interactedwith mTOR but inhibited by Akt3 siRNA by Western blots; (28 e)illustrates RA induces temporal expression of mTOR by Western blots; (28f) illustrates Akt3 siRNA inhibited the RA-induced phosphorylation ofmTOR; (28 g) illustrates mTOR directly interacted with 4EBP1 (4 hr); (28h) illustrates hTS cells treated by RA (4 hr) with or withoutpreincubation of mTOR siRNA or 4EBP1 siRNA were analyzed by Westernblots for expressions of mTOR, 4EBP1, eIF4E, and eIF4B; (28 i)illustrates eIF4E siRNA inhibited RA-induced interaction (4 hr) betweenRXRα and Gα_(q/11) (upper) and between RARβ and Gβ (lower) by Westernblots.

FIG. 29: (29 a) illustrates that PI3K inhibitor suppressed theRA-induced expression of Akt isoforms, Akt1, 2, and 3 after overnighttreatment in hTS cells by RT-PCR; (29 b) Akt2 inhibitor inhibitedexpression of β-catenin mRNA by RT-PCR; (29 c) Akt3 siRNA suppressedexpression of mTOR by flow cytometry.

FIG. 30 illustrates CREB1 promotes transcription of TH: (30 a)illustrates that CREB1 directly interacted with Akt1 and β-catenin byWestern blots; (30 b) illustrates that Akt1 siRNA inhibited expressionof CREB1. β-actin: control; (30 c) illustrates that CREB1 targeted atpromoter of TH gene; (30 d) illustrates that CREB1 siRNA inhibitedexpression of TH by Western blots; (30 e) illustrates thatimmunofluorescence tissue analysis revealed co-expression of TH-FITC(blue color) and TH-Cy-3 (red color) in DA neurons (white arrow) in thetherapeutic SNC side at 12 weeks postimplantation of tNSCs in PD ratbrain (right panel). Amplified DA neuron in the normal side (left upper)and the therapeutic sides (left lower). Positive CREB1 stain was foundin the nucleous; (30 f) illustrates that histograms showing the relativemean intensities of TH and CREB1 expressed in DA neurons in the normal(left; n=86) and the therapeutic sides (right; n=114). Error bars:mean±SD; n: total cells counted; p<0.05: significant statistically.

FIG. 31 illustrates immunohistofluoresence analysis: TH(+) and NeuN(+)motor neurons (arrow) in the SNC of control (left upper). DecreasedTH(+) (arrow) at 1-week after 6-OHDA injury (right upper). Apparentreduction in TH(+) neurons with disarrangement of TH-positive neuralterminals (green granules), and various degenerative cavity formation(red explosive circle) at 6-week post-injury (left lower). Aftertransplantation, TH(+) neurons (arrow) at wall of the degenerativecavity (red explosive circle; insert) with TH(+) neural terminals (greencolor) projecting into the cavity (right lower).

FIG. 32 illustrates in vivo regeneration of TH(+) and GFAP(+) cells withless immunoresponses: (32 a) illustrates a number of TH(+) cells at 1-and 6-week reduced to 48% and 13% in the lesioned SNC (dark grey) and78% and 4% in the lesioned striatum (light grey), respectively,post-injury. After transplantation, TH(+) cells re-grew up to 67% and73% in the lesioned SNC and striatum, respectively (right panel). Dataanalyzed by the software Tissuequest 2.0 (TissueGnostics Gmbh, Vienna,Austria); (32 b) illustrates regeneration of dopaminergic neurons in thelesioned SNC (lower panel) with amplification (left upper, insert a)compared with the intact side (right upper, insert b); (32 c)illustrates transplantation of tNSCs at 12 weeks yielded 78.4±8.3%(mean±SEM; n=4) of recovery rate in TH-positive neurons (arrow) in thelesioned SNC compared to the intact side; (32 d) illustratesdegeneration of TH-FITC(+) and GFAP-Cy-3(+) Wilson's pencils (blankarrow) at 6-week post-injury in the lesioned striatum (left column). At12 weeks postimplantation (right column), several GFAP(+) cells (arrow)appeared inside the fine fibers of re-established Wilson's pencils(blank arrow); (32 e) illustrates immunohistofluorescence imaginganalysis, cells were counted in the gate (left scatter plots) determinedby the location of cell size (8-10 μm in diameter) and its correspondingintensity of GFAP-Cy-3. Gate (red scatter plot): glial cells counted;black scatter plots: exclusive cells with bizarre size; blue scatterplots: cells with abnormal GFAP intensity. In the striatum, the GFAP(+)cells were 65.5% in the lesioned side before treatment and became 93.9%after cell therapy compared to the intact side (right panel); (32 f)illustrates hTS cells implantation into the SCID mice raised only minorimmunoreactions and without tumorigenesis observed. Myxoid-like bizarrecells (black arrow), muscle fibers (blank arrow), and needle track (NT).

FIG. 33 illustrates the qualification of TH(+) cells in the SNC beforeand after cell therapy, using the coefficient of determination betweenTH-FITC and NeuN-Cy-3, measured by immunohistofluorescent scatter plotsin the chronic PD rats. (33 left upper) illustrates normal SNC: R²=0.72;(33 right upper) illustrates SNC by 6-OHDA damage (1-week): R²=0.77; (33left lower) illustrates SNC by 6-OHDA damage (6-week): R²=0.25; (33right lower) SNC after tNSCs transplantation (12-week): R²=0.66. Resultsshown represent the average of 2 rats.

DETAILED DESCRIPTION OF THE INVENTION

Neural tissue-derived stem cells, phenotype-specified progenitor cellsderived from pluripotential embryonic stem cells (ESC), and neural cellsderived from various transdifferentiated non-neural stem cells have allbeen investigated in preclinical studies for their ability to generateneurons and glia, and the use of neural stem cells in clinical trialshas been described. Though embryonic stem (ES) cells have shownpotential as cell therapeutics, Bjorklund, L. M., et al. Proc. Nat.Acad. Sci. 2002, 99, 2344-49, access to such therapies is limited andassociated with ethical concerns.

Stem cells possess the capacity for self-renewal and to producecommitted progenitors including neural stem cells. Reubinoff B. E. etal., Nat. Biotech. 2001, 19, 1134-1140.

Provided herein are isolated neural stem cells that are derived fromtrophoblast tissue. Further provided herein are isolated neural stemcells (tNSCs) that are robust and survive several passages in cellculture and also possess characteristics of pluripotency and immuneprivilege. In one embodiment described herein, a method is described forinduction of dopaminergic neurons from tNSCs derived from humantrophoblast stem (hTS) cells. Further provided herein are methods thatallow for survival and growth of the grafted tNSCs into dopaminergicneurons, and methods for assessment of recovery of impaired behaviors toachieve results with reduced variability compared to current therapeuticregimens.

Also provided herein are isolated neural stem cells derived from hTScells that are cultured without using mouse embryonic feeder cells,circumventing problematic contaminations. Provided herein are methodsfor generation of hTS cell-derived tNSCs efficiently and reproducibly,leading to a uniformly mixed subset of populations that isdistinguishable from the other methods used to induce dopaminergicneurons from cells of other origins. Provided herein are methods fortransplantation of the dopaminergic tNSCs into the brain as a cellsuspension thereby circumventing uneven growth that is associated withtissue grafts.

Provided herein are methods of modulating a stem cell with an inductiondrug to differentiate into a cell with neuronal characteristics. In oneembodiment the induction drug modulates the expression or activity ofmodulating one or more proteins in the stem cell. In one embodiment oneof the one or more proteins is Wnt2B, Fzd6, Dvl3, FRAT1, GSK3β, HDAC6,β-catenin, Gα_(q/11), GP, RXRα, RARβ, GLuR1, PI3K, AKt1, AKt2, AKt3,mTOR, EIF4EBP, CREB1, TH (tyrosine hydroxylase), PLC-β, PIP2, CaMKII,EIF4B, parkin, SNCA, tublin, calcineurin, CRMP-2, NFAT1, importin, LEF1,Pitx2, MEF2A, or EP300. In one embodiment the stem cell can be atrophoblast, embryonic or induced progenitor stem cell. In oneembodiment the cell with neuronal characteristics is a NSC, dopamineproducing cell, dopaminergic neuron, unipolar neuron, bipolar neuron,multipolar neuron, pyramidal cell, Purkinje cell, and anterior horncell, basket cell, betz cell, Renshaw cell, granule cell, or mediumspiny cell.

Also provided herein are methods of modulating a stem cell with aninduction drug to differentiate into a cell with reduced immunogenicity.In one embodiment the induction drug modulates the expression oractivity of modulating one or more proteins in the stem cell. In oneembodiment one of the one or more proteins is Wnt2B, Fzd6, Dvl3, FRAT1,GSK3β, HDAC6, β-catenin, Gα_(q/11), Gβ, RXRα, RARβ, GLuR1, PI3K, AKt1,AKt2, AKt3, mTOR, EIF4EBP, CREB1, TH (tyrosine hydroxylase), PLC-β,PIP2, CaMKII, EIF4B, parkin, SNCA, tublin, calcineurin, CRMP-2, NFAT1,importin, LEF1, Pitx2, MEF2A, or EP300. In one embodiment the stem cellcan be a trophoblast, embryonic or induced progenitor stem cell. In oneembodiment the cell with reduced immunogenicity does not induce animmune response or can inhibit an immune response by a T cell, B cell,macrophage, microglia cell, mast cell, or natural killer (NK) cell.

Human Trophoblast Stem Cells (hTS Cells)

Human fallopian tubes are the site of fertilization and the common siteof ectopic pregnancies in women, where several biological events takeplace such as the distinction between inner cell mass (ICM) andtrophectoderm and the switch from totipotency to pluripotency with themajor epigenetic changes. These observations provide support forfallopian tubes as a niche reservoir for harvestingblastocyst-associated stem cells at the preimplantation stage. Ectopicpregnancy accounts for 1 to 2% of all pregnancies in industrializedcountries and are much higher in developing countries. Given theshortage in availability of human embryonic stem cell (hES cells) andfetal brain tissue, described herein is the use of human trophoblastcells (hTS cells) derived from ectopic pregnancy as a substitution forscarcely available hES cells for generation of progenitor cells.

In one embodiment, the human trophoblast cells derived from ectopicpregnancies do not involve the destruction of a human embryo. In anotherembodiment, the human trophoblast cells derived from ectopic pregnanciesdo not involve the destruction of a viable human embryo. In anotherembodiment, the human trophoblast cells are derived from trophoblasttissue associated with non-viable ectopic pregnancies. In anotherembodiment, the ectopic pregnancy cannot be saved. In anotherembodiment, the ectopic pregnancy would not lead to a viable humanembryo. In another embodiment, the ectopic pregnancy threatens the lifeof the mother. In another embodiment, the ectopic pregnancy is tubal,abdominal, ovarian or cervical.

During blastocyst development, ICM contact per se or its deriveddiffusible ‘inducer’ triggers a high rate of cell proliferation in thepolar trophectoderm, leading to cell movement toward the mural regionthroughout the blastocyst stage and can continue even after thedistinction of the trophectoderm from the ICM. The mural trophectodermcells overlaying the ICM are able to retain a ‘cell memory’ of ICM.Normally, at the beginning of implantation the mural cells opposite theICM cease division because of the mechanical constraints from theuterine endometrium. However, no such constraints exist in the fallopiantubes, resulting in the continuing division of polar trophectoderm cellsto form extraembryonic ectoderm (ExE) in the stagnated blastocyst of anectopic pregnancy. In one embodiment, the ExE-derived TS cells exist forat least a 4-day window in a proliferation state, depending on theinterplay of ICM-secreted fibroblast growth factor 4 (FGF4) and itsreceptor fibroblast growth factor receptor 2 (Fgfr2). In anotherembodiment, the ExE-derived TS cells exist for at least a 1-day, atleast a 2-day, at least a 3-day, at least a 4-day, at least a 5-day, atleast a 6-day, at least a 7-day, at least a 8-day, at least a 9-day, atleast a 10-day, at least a 11-day, at least a 12-day, at least a 13-day,at least a 14-day, at least a 15-day, at least a 16-day, at least a17-day, at least a 18-day, at least a 19-day, at least a 20-day windowin a proliferation state. Until clinical intervention occurs, thesecellular processes can yield an indefinite number of hTS cells in thepreimplantation embryos; such cells retaining cell memory from ICM,reflected by the expression of ICM-related genes.

One aspect described herein are hTS cells and chorionic cytotrophoblastsbefore uterine implantation. In one embodiment, hTS cells possess bothspecific genes of inner cell mass (ICM) (Oct4, Nanog, Sox2, FGF4) andtrophectoderm (Cdx2, Fgfr-2, Eomes, BMP4) (FIG. 1a ) and expresscomponents of all three primary germ layers (FIG. 7). In anotherembodiment, the hTS cells express hES cell-related surface markers suchas specific stage embryonic antigen (SSEA)-1, -3 and -4 (FIG. 1b ) andmesenchymal stem cell-related markers (CD 44, CD90, CK7 and Vimentin),while hematopoietic stem cell markers (CD34, CD45, α6-integrin,E-cadherin, and L-selectin) were not expressed (FIG. 8). In oneembodiment, hTS cells could be differentiated into a variety of specificcell phenotypes of three primary germ layers upon induction (FIG. 9).Transplantation of hTS cells into the male severe combined immunedeficiency (SCID) mice subcutaneously caused only minor chimericreaction at 6-8 weeks postimplantation histologically (FIG. 10). In oneembodiment, chromosome analysis showed that hTS cells did not change thepatterns of karyotypes (46, XY) (FIG. 11). In another embodiment, thecell lifespan was not significantly shortened in telomere length between3rd and 7th passage in culture (FIG. 1c ).

One aspect provided herein describes the distinction between hTS cellsand placenta derived mesenchymal stem (PDMS) cells, using theAffymetrix™ platform to interrogate the GeneChip Human Genome U133 plus2.0 GeneChip for a global gene comparison between hTS cells and PDMScells. In one embodiment, the hTS cells exhibited about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65% about 70%, or about 75% less geneexpression than that in PDMS cells. In another embodiment, the hTS cellsexhibited total 2,140 genes (fold change >2-fold) which is about 40%less than that in PDMS cells (3,730 genes) (FIG. 1d ). In oneembodiment, the gene intensity distribution of hTS cells displayed ahomogenous pattern distinct from that in PDMS cells. In anotherembodiment, the hTS cells represent a distinct group of cytotrophoblastsat a stage of pre-implantation, whereby they possess molecular portraitsof inner cell mass (ICM) and/or trophectoderm. In another embodiment,the hTS cells exhibit characteristics of pluripotency and self-renewalsimilar to that of hES cells.

Withdrawal of LIF Mediates Overexpression of Nanog in hTS Cells

Cytotrophoblasts are the precursors of syncytiotrophoblasts in humans(Benirschke, K., Kaufmann, P. in Pathology of the human placenta, 39-51Spring-Verlag New York Inc., 1990). A zone of trophoblast specificationis established when the embryo is a morula, reflecting a distinctcombination of transcription factors in that zone of cells and theinfluence of various environmental cues and growth factors on them.

Much evidence indicates that naive pluripotency of early epiblast andauthentic ES cells are dependent on the action of three transcriptionalorganizers, Oct4, sex determining region Y-box 2 (Sox2), and Nanog(Chambers I., et al., Oncogene, 23:7150-7160 (2004); Niwa H.Development, 134:635-646 (2007)). ES cells maintain pluripotency througha complex interplay of different signaling pathways and transcriptionfactors, including leukemia inhibitory factor (LIF), Nanog, Sox2, andoctamer-binding transcription factor 3 and 4 (Oct3/4). The transcriptionfactor Nanog plays a key role in maintaining the pluripotency of mouseand human ES cells, while LIF works in concert with Oct4 and Nanog tosupport pluripotency and self-renewal (Cavaleri, F. et al. Cell 113,551-552 (2003)).

LIF, an interleukin-6 class cytokine, affects cell growth anddifferentiation. LIF binds to leukemia inhibitory factor receptor alpha(LIFR-alpha), which forms a heterodimeric receptor complex with membraneglycoprotein 130 (GP130) common receptor. The binding of LIF leads tothe activation of janus kinase (JAK)/signal transducer and activator oftranscription (STAT) signaling pathways as well as mitogen-activatedprotein kinase (MAPK) pathways. LIF is normally expressed in thetrophectoderm of developing embryo. LIF is thought to play a role inmaintaining undifferentiated state. Removal of LIF from a stem cellculture usually leads to differentiation of the cultured stem cell. LIFalso affects the expression of Nanog, a gene known to play a crucialrole in stem cell maintenance.

Normally, a pleiotropic cytokine leukemia inhibitory factor (LIF) isexpressed at a higher concentration in the fallopian tubes than in theendometrium, showing a gradient reduction from the ampulla to theisthmic segment (FIG. 1g ). While in ectopic pregnancy, LIF levels canincrease 2 to 4-fold in the fallopian tube (Wånggren, K., et al., Mol.Hum. Reprod. 2007, 13, 391-397). Functionally, LIF can integrate othersignals to activate pluripotent transcription factors, for example, Oct4and Nanog, to maintain pluripotency and self-renewal in mouse embryonicstem (mES) cells. On withdrawal of LIF, cell proliferation continues buta caudal-related homeobox transcription factor Cdx2 is activated,triggering for trophectoderm differentiation in embryonic stem (ES)cells.

In one embodiment, a method is described to determine how hTS cellsmaintain characteristics of pluripotency and self-renewal. In oneembodiment, the association of LIF with pluripotent transcriptionfactors (e.g., factors described in Smith, A. G., et al., Nature 336,688-690 (1998), Williams, R. L., et al., Nature 336, 684-687, (1998),Cavaleri, F. et al., Cell 113, 551-552 (2003); Chambers I., et al.,Cell, 2003; 113:643-655, Boiani, L. A. et al., Nature Rev. Mol. CellBiol. 6, 872-884 (2005)) was examined in hTS cells.

hTS cells were obtained from women who had suffered tubal ectopicpregnancies at 5-8 weeks of gestation and characterized as a distinctpopulation of cytotrophoblasts, possessing specific genetic markers(e.g., markers described in Adjaye, J., et al., Stem Cells, 2005, 23,1514-1525) of ICM-derived human embryonic stem (hES) cells andtrophectoderm (FIG. 1a ).

Provided herein, in one embodiment, is a method to affect hTS celldifferentiation by modulating the exposure of said cell to LIF. Forexample, hTS cells are divided into three groups and exposed todifferent concentrations of LIF. In one embodiment, the concentration ofLIF is about 1000, about 750, about 600, about 550, about 525, about500, about 450, about 400, about 350, about 300, about 250, about 200,about 150, about 125, about 100, about 75, about 50, or about 25Units/mL. In another embodiment, the concentrations of LIF are 500, 250,and 125 Units/mL. In one embodiment, the concentration of LIF is 500Units/mL. In another embodiment, the concentration of LIF is 250Units/mL. In another embodiment, the concentration of LIF is 125Units/mL.

In one embodiment, the hTS cells are exposed to different concentrationsof LIF for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or days. In anotherembodiment, the hTS cells are exposed to different concentrations of LIFfor 3, 6, 12, 18, 24, 30, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144,156, 168, 180, 192, 204, 216, 228, 240, or 252 hours. In anotherembodiment, the hTS cells are exposed to different concentrations of LIFfor about 1 to 30, about 1 to 28, about 1 to 26, about 1 to 24, about 1to 22, about 1 to 20, about 1 to 18, about 1 to 15, about 1 to 13, about1 to 10, about 1 to 9, about 1 to 8, about 1 to 7, about 1 to 6, about 1to 5, about 1 to 4, or about 1 to 2 days. In another embodiment, the hTScells are exposed to different concentrations of LIF for 3 days.

One aspect described herein is that lower concentrations of LIF changingthe expression of certain genes, which include but are not limited toOct4, Sox2, Cdx2, and Nanog. Another embodiment demonstrates thatwithdrawal of LIF and/or lower concentrations of LIF suppresses Oct4 andSox2 expressions, and in contrast, promotes Cdx2 and Nanog by RT-PCR(FIG. 1e ). In one embodiment, these phenomena were further confirmed byflow cytometric analysis, showing suppression of Oct4 and Sox2, in adose-dependent manner (FIG. 1f ).

In another embodiment, the relative expression of Oct4/Cdx2 ratioindicates cell fate in early embryonic differentiation. In anotherembodiment, the withdrawal and/or decrease of LIF exposure leads to adecrease in Oct4 expression. In another embodiment, the withdrawaland/or decrease of LIF exposure promotes the expressions oftranscription factors Cdx2, Nanog, and Sox2 in a dose-dependent manner,which is consistent with quantitative PCR (qPCR) analyses.

Another aspect is described herein is a high Oct4/Cdx2 ratio at theampulla with a gradient reduction toward the isthmic segment in hTScells (FIG. 1g ) compatible with the trend of LIF levels in thefallopian tubes, thereby implying a cell fate choice toward hES cells.In one embodiment, upregulation of relative Nanog/Cdx2 ratio (2-fold)further enforces pluripotency in the cell. In one embodiment,upregulation of relative Nanog/Cdx2 ratio (2-fold) maintainspluripotency in the hTS cell. In another embodiment, the Sox2/Cdx2expression ratio does not change for the hTS cells to maintainpluripotency. In another embodiment, Cdx2 overexpression is favorablefor the hTS cells to maintain a trophoblastic phenotype.

One embodiment described herein is a method to examine the relationshipbetween Nanog and Cdx2 in hTS cells. In another embodiment, knockoutstudies of both Nanog and Cdx2 by using siRNA promotes Cdx2 and Nanogexpressions, respectively (FIG. 1h ), supporting the reciprocalrelationship between Nanog and Cdx2 in hTS cells similar to that of Oct4and Cdx2 in ES cells for cell fate choice (Niwa, H., et al., Cell 123,917-929). In another embodiment, overexpression of Nanog in combinationwith elevated Nanog/Cdx2 ratio compensates for the decreased Oct4/Cdx2ratio and is sufficient for the maintenance of pluripotency and/orrenewal which determines cell differentiation fate in hTS cells.

One aspect described herein shows that overexpression of Nanog uponwithdrawal of LIF is at least one factor that plays a role inmaintaining the pluripotency of hTS cells.

Retinoic Acid (RA) and Related Pathways

Retinoic acid (RA), a derivative of vitamin A, plays a role in ES celldifferentiation and embryogenesis. In ES cells, RA acts by binding toits nuclear receptors and inducing transcription of specific targetgenes to generate a number of different cell types. In one embodiment,induction with RA enables an hTS cell-derived tNSCs to sustain a stablyundifferentiated state with specific patterning.

In one embodiment, treating hTS cells with all trans-retinoic acid (RA)produces neural stem cells suitable for implantation into a rat diseasemodel (e.g., Parkinson's disease model). In another embodiment,withdrawal and/or a decrease of LIF exposure in hTS cells mediatesoverexpression of Nanog, which is responsible for the pluripotency andmaintenance of self-renewal of hTS cells. Also described herein arecertain molecular pathways that allow RA induced hTS cells todifferentiate into neural stem cells including pathways that play a rolein reversible epithelial-mesenchymal transition (EMT), bonemorphogenetic protein (BMP) and Wnt signaling pathway cross-talk, andtriggering the target gene Pitx2 for neural stem cells formation.Accordingly, one embodiment describes the use of modulators ofRA-related pathways for generation of neural stem cells from hTS cells.

RA Induces a Uniform Complex of NSC Subtypes

In one embodiment, hTS cells are induced to produce neural stem cells.In one embodiment, the hTS cells are exposed to or treated with aninducing agent. In one embodiment, an inducing agent includes but is notlimited to retinoic acid, nerve growth factor, basic fibroblast growthfactor, neurotropins (e.g., neurotropin 3) and/or combinations thereof.Additional exemplary inducing agents include, but are not limited to:erythropoietin (EPO), brain derived neurotrophic factor (BDNF),wingless-type MMTV integration site (Wnt) proteins (e.g., Wnt3a),transforming growth factor alpha (TGFα), transforming growth factor beta(TGFβ), bone morphogenetic proteins (BMPs), thyroid hormone (TH,including both the T3 and T4 forms), thyroid stimulating hormone (TSH),thyroid releasing hormone (TRH), hedgehog proteins (e.g., sonichedgehog), platelet derived growth factor (PDGF), cyclic AMP, pituitaryadenylate cyclase activating polypeptide (PACAP), follicle-stimulatinghormone (FSH), growth hormone (GH), insulin-like growth factors (IGFs,e.g., IGF-1), growth hormone releasing hormone (GHRH), prolactin (PRL),prolactin releasing peptide (PRP), fibroblast growth factor (FGF),estrogen, serotonin, epidermal growth factor (EGF), gonadotropinreleasing hormone (GnRH), ciliary neurotrophic factor (CNTF), leukemiainhibitory factor (LIF), granulocyte colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), vascularendothelial growth factor (VEGF), luteinizing hormone (LH), humanchorionic gonadotropin (hCG), pheromones (e.g.,2-sec-butyl-4,5-dihydrothiazole, 2,3-dehydro-exo-brevicomin, alpha andbeta farnesenes, 6-hydroxy-6-methyl-3-heptanone, 2-heptanone,trans-5-hepten-2-one, trans-4-hepten-2-one, n-pentyl acetate,cis-2-penten-1-yl-acetate, 2,5-dimethylpyrazine, dodecyl propionate, and(Z)-7-dodecen-1-yl acetate), and/or combinations thereof. In anotherembodiment, the inducing agent is an analog or variant that has theactivity of the native inducing agent.

By way of non-limiting example, retinoic acid is used to chemicallyinduce hTS cells. The pleiotropic factor all-trans retinoic acid (RA)plays in vivo functions in neural differentiation, patterning and motoraxon outgrowth via multiple pathways, including but not limited toRA/RARs/RXRs signaling, Wnt signaling and ERK pathway in ES cells(Maden, M. Nat. Rev. Neuroscience 8, 755-765 (2007), Lu J, et al., BMCCell Biol. 2009, 10: 57, Wichterle H, et al., Cell. 2002; 110:385-397).RA induces the expression of tyrosine hydroxylase (TH), the hallmarkenzyme of dopaminergic neurons, and the neurite formation in mES cells(Wichterle H, et al., Cell. 2002; 110:385-397), hES cells (Li, L. et al.Stem Cells 22, 448-456 (2004)) and adult neurogenesis (Jacobs S, et al.,Proc Natl Acad Sci 2006, 103(10):3902-7).

In one embodiment, a method is described to determine the fate of hTScells treated with RA. In another embodiment, the hTS cells are treatedwith 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20,25, 30, 35, 40, 45, 50, 55, 60, or 65 μM of RA. In another embodiment,the hTS cells are treated with about 0.5-75, about 1-65, about 1-60,about 1-50, about 1-55, about 1-50, about 1-40, about 1-35, about 1-30,about 1-25, about 1-20, about 1-15, about 1-13, about 1-10, about 2-10,about 5-10, or about 8-10 μM of RA. In another embodiment, the hTS cellsare treated with 10 μM of RA.

In one embodiment, the hTS cells are exposed to RA for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40days. In another embodiment, the hTS cells are exposed to RA for 3, 6,12, 18, 24, 30, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168,180, 192, 204, 216, 228, 240, or 252 hours. In another embodiment, thehTS cells are exposed to RA for about 1 to 20, about 1 to 18, about 1 to15, about 1 to 13, about 1 to 10, about 1 to 9, about 1 to 8, about 1 to7, about 1 to 6, about 1 to 5, about 1 to 4, or about 1 to 2 days. Inanother embodiment, the hTS cells are exposed to RA for differentdurations: 1, 2, 3, 4, 5, 6, 7, or 8 days each. In another embodiment,the hTS cells are exposed to RA for 1 day. In another embodiment, thehTS cells are exposed to RA for 2 days. In another embodiment, the hTScells are exposed to RA for 3 days. In another embodiment, the hTS cellsare exposed to RA for 4 day. In another embodiment, the hTS cells areexposed to RA for 5 days. In another embodiment, the hTS cells areexposed to RA for 6 day. In another embodiment, the hTS cells areexposed to RA for 7 days. In another embodiment, the hTS cells areexposed to RA for 8 day.

In one embodiment, RA induces hTS cells differentiation into a varietyof phenotypical neural cells, which include but are not limited to glialrestricted precursors (GRP), neuronal restricted precursors (NRP),multipotent neural stem (MNS) cells, astrocytes (AST) and undefinedtrophoblast giant cells (TGC), expressing neural stem cell marker nestinimmunocytochemically (FIG. 2a ). In another embodiment, a similar ratioin distribution of mixed RA-induced neural progenitors results over 1 to5-day RA-induction periods. In another embodiment, the celldifferentiation becomes undefined trophoblast giant cells over a 7-dayRA treatment.

Accordingly, provided herein, in one embodiment, are RA-induced neuralstem cells derived from hTS cells. In another embodiment, the RAinduction period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, or 40 days. In another embodiment, theRA induction period is 3, 6, 12, 18, 24, 30, 36, 48, 60, 72, 84, 96,108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, or 252hours. In another embodiment, the RA induction period is about 1 to 20,about 1 to 18, about 1 to 15, about 1 to 13, about 1 to 10, about 1 to9, about 1 to 8, about 1 to 7, about 1 to 6, about 1 to 5, about 1 to 4,or about 1 to 2 days. In one embodiment, the RA induction period is fromabout one day to about 7 days. In another embodiment, the RA inductionperiod is one day. In another embodiment, the RA induction period is 2days. In another embodiment, the RA induction period is 3 days. Inanother embodiment, the RA induction period is 4 days. In oneembodiment, the RA induction period is 5 days. In one embodiment, the RAinduction period is 6 days. In another embodiment, the RA inductionperiod is 7 days. In another embodiment, the RA induction period is 24hours. In one embodiment, the RA induction period is 12 hours. Inanother embodiment, the RA induction period is 1 hour to 24 hours.

Described herein, in one embodiment, is a tNSC that expresses at leastone neural stem cell gene and marker. In another embodiment, the tNSCexpresses at least two, at least three, at least four or at least fiveneural stem cell genes. In another embodiment, the tNSC expresses atleast two, at least three, at least four or at least five neural stemcell markers. Non-limiting examples of neural stem cell genes andmarkers include nestin, neurofilament, Ngn-3, MAP-2, Neo-D, CD133 andOct4 (FIG. 2b ). In one embodiment, the tNSCs also express RA receptorgenes, which include but are not limited to RARβ, RXRα, and RXRβ,cellular retinoic acid binding protein (CRABP)-2, cellular retinolbinding protein (CRBP)-1 and specifically, RA-synthesizing enzymesRALDH-2 and -3 which were found to be absent in ES cells.

Accordingly, one embodiment describes the use of expressed neural stemcell genes and markers, including nestin, neurofilament, Ngn-3, MAP-2,Neo-D, CD133 and Oct4, RA receptor genes such as RARβ, RXRα and RXRβ,CRABP-2, CRBP-1, RA-synthesizing enzymes RALDH-2 and -3 or the like,and/or modulators thereof, to facilitate the differentiation capacity oftNSCs. In one embodiment, both 3- and 5-day RA-induced hTS cells sustainneural stem cells markers in a similar ratio, including nestin, GFAP andneurofilament protein (FIG. 2c ). In another embodiment, these tNSCsexpressed tyrosine hydroxylase (TH) and 5-hydroxytryptamine (5-HT)immunocytochemically (FIG. 2d ), implying their capacity to bedifferentiated into dopaminergic as well as serotonergic neurons.Another embodiment described herein is the differentiation of tNSCs todopaminergic neurons and serotonergic neurons.

Further provided herein are tNSCs that consist of uniformly mixedneuroepithelial progenitor cells sustainable in a steady-state,genetically and phenotypically, in cell culture. This consistency inproduct is a desirable characteristic for any treatment regimencomprising stem cell-based therapy.

Association Between LIF and RA in Respect of Nanog Expression

In early embryonic development, tNSCs typically express RALDH-2. Oneembodiment described herein is a method to evaluate how LIF affects theRA-induced neurogenesis in hTS cells. The ability of LIF to inhibitRA-induced neuronal differentiation in mouse ES (mES) cells, renderstransplantation more difficult (Martín-Ibáñez R, et al., J. Neuron. Res.85, 2686-2710 (2007), Bain G, et al., Dev Biol 168: 342-357). Otherreports claim a positive role of LIF in the differentiation of ES cellsinto neurons (Tropepe V, Neuron 2001, 30: 65-78).

In one embodiment, a method is described to evaluate the associationbetween LIF and RA in respect of Nanog expression in hTS cells. Inanother embodiment, tNSCs are treated with LIF and subjected formeasurement of Nanog expression by flow cytometry (FIG. 18a ). In oneembodiment, the tNSCs are treated with about 1000, about 750, about 600,about 550, about 525, about 500, about 450, about 400, about 350, about300, about 250, about 200, about 150, about 125, about 100, about 75,about 50, or about 25 Units/mL of LIF. In another embodiment, the tNSCsare treated with 1-1000, 1-500, 1-450, 1-400, 1-350, 1-300, 1-250,1-200, 1-150, 1-125, 1-100, 1-75, or 1-50 Units/mL of LIF. In anotherembodiment, the tNSCs are treated with 500, 250, and/or 125 Units/mL ofLIF.

In one embodiment, the hTS cells are exposed to LIF for 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 days. In another embodiment, the hTS cells are exposedto LIF overnight. In another embodiment, the hTS cells are exposed toLIF for 3, 6, 12, 15, 18, 22, 24, 30, 36, 48, 60, 72, 84, 96, 108, 120,132, 144, 156, 168, 180, 192, 204, 216, 228, 240, or 252 hours. Inanother embodiment, the hTS cells are exposed to LIF for about 1 to 20,about 1 to 18, about 1 to 15, about 1 to 13, about 1 to 10, about 1 to9, about 1 to 8, about 1 to 7, about 1 to 6, about 1 to 5, about 1 to 4,or about 1 to 2 days.

In one embodiment, treatment of hTS cells with RA induces Nanogoverexpression. In another embodiment, LIF suppresses the RA-inducedNanog in a dose-dependent manner. In another embodiment, LIF exerts aninhibitory action on tNSC development.

One aspect described herein is that LIF interplays with RA on neuraldifferentiation of ES cells. In one embodiment, LIF influences theeffect of RA on the pluripotency in hTS cells. Results showed that RAinduced overexpression of Nanog and Oct4 but not Cdx2 and Sox2 in hTScells (FIG. 18b ). In the isthmus region of the brain, Nanog expressionwas observed in 62.5% in LIF-induced cells (FIG. 1F, left and rightpanel) but only 26.9% in RA-induced cells (FIG. 18b ). It was alsoobserved that a higher level of LIF generally repressed the RA-inducedNanog and withdrawal of LIF significantly enhanced the RA-induced Nanogexpression (FIG. 18a ). These results indicated that as hTS cells movetowards the isthmus. In one embodiment, RA maintains cellularpluripotency by Nanog expression.

In one embodiment, implantation of tNSCs in an RA-enrichedmicroenvironment facilitates the continuous proliferation of stem cellsin vivo. In another embodiment, the tNSCs are implanted into the brain.In another embodiment, the tNSCs are implanted or injected into thehippocampus, cerebral cortex, striatum, septum, diencephalon,mesencephalon, hindbrain, or spinal cord basal ganglia. In anotherembodiment, the tNSCs are implanted into the striatum of brain. Inanother embodiment, the tNSCs are implanted or injected into any part ofthe central nervous system. In another embodiment, the tNSCs areimplanted or injected into the nerve terminal area of the cells thatdegenerate in the particular neurodegenerative disorder. In anotherembodiment, the tNSCs are implanted or injected into midbrain insubstantia nigra pars compact. In another embodiment, the tNSCs areimplanted or injected into the nerve terminal area in the forebrain. Inanother embodiment, the tNSCs are implanted or injected into theventricular system. In another embodiment, the tNSCs are implanted orinjected into the lateral ventricle.

G Protein Signaling in the Maintenance of Multipotency in tNSCs

Another aspect described herein is a method to investigate how tNSCssustain their multipotency status. In one embodiment, RA induces c-SrcmRNA expression peaks at about 15 min (FIG. 3a ). Another embodimentdescribed herein evaluates the GPCR signaling pathway based on RAstimulated expression of RXRα, c-Src and RARβ by Western blot analysis(FIG. 3b ). In one embodiment, RA promotes both Gα_(q/11) and Gβexpressions in 30 min. In another embodiment, analysis ofimmunoprecipitation (IP) assays demonstrate that RA induces directbinding between RXRα and RARβ; however, this interaction is blocked byc-Src inhibitor PP1 analog, indicating that c-Src is involved in betweenRXRα and RARβ to form a scaffolding protein complex (FIG. 3c ).

By immunoprecipitation (IP) analysis (FIG. 3d ), we observed that RXRαdisplays binding interactions with Gα_(q/11) while RARβ shows bindinginteractions with Gβ independently. These results are compatible withthe ‘pull and push’ model of GPCR-G protein signaling (Tsai et al., “Theubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 fordegradation. Nat. Med. 13, 1504-1509, (2007)).

In one embodiment, the heterodimeric pair of RARs and RXRs plays therole of ligand-activated transcription factors in the nucleus andendogenous cell surface signal molecules. The constitutively activatedRXRα breaks up the receptor conformations and recruits c-Src to interactwith and/or activate the associated Gα_(q/11). In one instance, thisnon-genomic RA signal transduction assists in the interpretation ofnon-retinoic acid-response element (RARE)-mediated gene expression(Maden, M., Nat. Rev. Neuroscience 8, 755-765 (2007)).

Accordingly provided herein are strategies for preventing cellularovergrowth before and after transplantation of neural stem cellsprovided herein. One embodiment describes the use of agents thatmodulate RA-related pathways thereby preventing, and/or reducing and/oralleviating overgrowth and/or graft rejection.

Proto-Oncogene Tyrosine-Protein Kinase (Src) and Nanog

c-Src maintain ES cells at an undifferentiated state (Annerén C. et al.,J Biol Chem. 279, 590-598 (2004)). Nanog and Stat3 bind synergisticallyto activate Stat3-dependent promoters (Tones J., et al., Nat Cell Biol.10, 194-201 (2008)). In one embodiment, c-Src induces signal transducerand activator of transcription 3 (Stat3) phosphorylation at Tyr705 siteand this action is blocked by c-Src inhibitor protein phosphatase 1(PP-1) analog, thereby linking the association between c-Src and Stat3molecules (FIG. 3f ). In another embodiment, Stat3 acts directly on theNanog promoter (FIG. 3g ). In another embodiment, Stat3 does not actdirectly on the Nanog promoter. In another embodiment, RXRα actsdirectly on the Nanog promoter. In another embodiment, RXRα does not actdirectly on the Nanog promoter. In another embodiment, RARβ actsdirectly on the Nanog promoter. In another embodiment, RARβ does not actdirectly on the Nanog promoter. In another embodiment, RA inducesoverexpression of c-Src, pStat3 (FIG. 3e ) and Nanog (FIG. 1e ) in hTScells. In another embodiment, both RXRα and RARβ play a transductionalrole in response to RA via GPCR-G protein signaling.

Described herein, in one embodiment, is a method to maintainmultipotency in tNSCs, the method comprising activating thec-Src/Stat3/Nanog transcription pathway. In another embodiment,interaction of c-Src and Gα_(q/11) activates of c-Src/Stat3/Nanogpathway. To further verify the direct interaction between RXRα andGα_(q/11) by imaging study, double immunogold fluorescence transmissionelectron microscopy (IEM) was utilized. RA induced binding interactionbetween the small gold particle-labeled RXRα (6 μm) and the large goldparticle-labeled G_(αq/11) (20 μm) at the plasma membrane (FIG. 4). Bydynamic confocal immunofluorescence microscopy, both immunostained RXRαand G_(αq/11) appeared primarily in a homogenous feature in eithercytoplasm or nucleus (FIG. 4, upper panel). By treatment with RA for 5min, the cytosolic RXRα intensity increased at the peri-nuclear regionswhile the nuclear one decreased (FIG. 4, first column), indicating acytosolic translocation after stimulation. The nuclear RXRα intensitybecame prominent at 15 min, while the cytosolic one decreased (FIG. 3a).

In one embodiment, an increase of activity in a cell nucleus maintains asteady-status in the cell. An apparent cytosolic translocation wasobserved again in 30 min. The compartmental changes of G_(αq/11)expression, on the other hand, were similar to that RXRα (FIG. 4, secondcolumn). In one embodiment, there was an apparent accumulation ofG_(αq/11) observed at the cell membrane at 30 min after stimulation. Inanother embodiment, RA enables promotion of both RXRα and G_(αq/11)synthesis and translocalization constitutively in hTS cells.

Accordingly, provided herein is the use of RA acting on hTS cells via Gprotein-coupled receptor (GPCR)-G proteins signaling at the plasmamembrane, which is distinguishable from genomic RA/RXRs/RARs pathways,for generation of tNSCs. As shown here, RA acts through Nanog and Oct4,but not Cdx2 and Sox2 pathways, in differentiating hTS cells into tNSCs.Also provided herein is the use of RA-induced Nanog activation for themaintenance of multipotency and self-renewal in tNSCs. Provided hereinis the use of RA activation of G protein-coupled receptor (GPCR)-Gprotein signaling, and concomitant activation of theRXRα/Gαq/11/c-Src/Stat3/Nanog pathway, for the maintenance ofmultipotency in tNSCs. Provided herein is the use of the heterodimers ofRXRα and RARβ functioning as signaling molecules at the plasma membranefor the maintenance of multipotency in tNSCs. Also provided herein isthe use of RA induced differentiation of hTS cells into neural stemcells (NSCs) by overexpression of Nanog for the maintenance ofpluripotency and renewal.

The tNSCs described herein express retinaldehyde dehydrogenase (RALDH)-2and -3 which aids neurogenesis. The presence of RALDHs and absence ofCD33 in the tNSCs described herein indicates that the tNSCs are superiorto hES cells in the differentiation into sensorimotor neurons.Accordingly provided herein is the use of tNSCs described herein forneurogenesis and/or regenerative medicine.

In the developing striatum and hippocampus, an increased Src kinaseactivity coincides with the peak period of neuronal differentiation andgrowth. However, RA can suppress phosphorylation of ribosomal S6 kinaseand its downstream eukaryotic Initiation factor 4B (eIF4B) by 24 hrincubation to cause growth arrest of many cell types. RA induces arapidly transient expression of c-Src mRNA peaking at 15 min (FIG. 3a ),followed by production of c-Src protein at 1 hr in hTS cells (FIG. 3e ).In one embodiment, c-Src mRNA contains an internal ribosome entry site.In another embodiment, RA transiently produces eIF4B peaking at 4 hr,but fading away at 24 hr (FIG. 20c ). This action was inhibited by usingeIF4B siRNA (FIG. 20d ). The involvement of mTOR/eIF4EBP1 signaling(mechanistic target of rapamycin/eukaryotic Initiation factor 4E bindingprotein 1) was excluded (FIG. 20b ). In another embodiment, RA activateseIF4B for subcellular mRNA localization to produce c-Src.

Active c-Src binds directly to Stat3 (signal transducer and activator oftranscription) (FIG. 20e ) by phosphorylation at site Tyr705 to produceprotein (FIG. 3e ). In one embodiment, this action is inhibited by usingc-Src siRNA (FIG. 20f ). In another embodiment, this action is inhibitedby a selective c-Src inhibitor PP-1 analog (FIG. 3f ). In anotherembodiment, a direct action of Stat3 on the Nanog gene promoter isobserved by chromatin immunoprecipitation (ChIP) assay (FIG. 3g ). Inanother embodiment, Nanog is produced in 4 hr (FIGS. 3f and 20f ), whichwas able to be blocked by using PP1 analog (FIG. 3f ) and Stat3 siRNA(FIG. 20g ).

Described herein, in one embodiment, is a method to maintainpluripotency of tNSCs comprising exposure of the cell an inducing agentto modulate the nongenomic eIF4B/c-Src/Stat3/Nanog signaling pathwaymediated c-Src subcellular mRNA localization (FIG. 20h ). In anotherembodiment, the inducing agent is RA.

RA and Wnt Signaling

Also provided herein is a method to induce hTS cells into neural stemcells. In one embodiment, the method comprises modulating theWnt2B/beta-catenin signaling pathway. In another embodiment, the methodcomprises modulating the RARs-Akt signaling pathway. In anotherembodiment, the method comprises modulating the Wnt2B/beta-catenin andRARs-Akt signaling pathways. In another embodiment, the hTS cells areinduced by treatment with retinoic acid (RA). In another embodiment, themethod to induce hTS cells into neural stem cells further comprisesactivating transcription factor Pitx2. In another embodiment, the methodto induce hTS cells into neural stem cells further comprises activatingtranscription factor netrin (NTN). In another embodiment, the method toinduce hTS cells into neural stem cells further comprises activatingtranscription factors Pitx2 and NTN. In another embodiment, the RAR andRXR exist as a heterodimer bound through its DNA-binding domain (DBD) tothe retinoic acid responsive element (RARE) DR-5. In another embodiment,corepressors bind to RAR and recruit histone deacetylase (HDAC) causingtranscriptional repression. In another embodiment, the method to inducehTS cells into neural stem cells further comprises activatingtranscription factors Pitx2 and NTN. In another embodiment, RA is addedto hTS cells and transcription is activated by RA binding to the RAR. Inanother embodiment, RAR binds to RA then recruits coactivators and HAT.

RA-mediated Wnt signaling pathway is a crucial contributor during adultneurogenesis and survival in vivo. Wnt proteins, present in the neuralstem cell microenvironment, are key regulators of cellular behavior inearly embryogenesis and can maintain neural stem cell potency. In adultneurogenesis, Wnt proteins bind to their receptor Frizzled (e.g., Fzd6)to transduce numerous signaling cascades, for example, by activating thebeta-catenin/LEF signaling for specific target genes.

Wnt signals are involved in cell cycle control and morphogenesis duringneurodevelopment. Among them, Wnt2B can inhibit differentiation ofretinal neurons and has been suggested to be a stem cells factor forNSCs using comparative integromics analysis. In one embodiment, Wnt2Bmodulates the expression of frizzled family receptor 6 (Fzd6). Inanother embodiment, Wnt2B induces the expression of Fzd6. In anotherembodiment, Fzd6 is overexpressed in the presence of Wnt2B. In oneembodiment, RA modulates a canonical Wnt2B/Fzd6/β-catenin signalingpathway for the dopaminergic differentiation in hTS cells. In oneembodiment, RA induces a canonical Wnt2B/Fzd6/β-catenin signalingpathway for the dopaminergic differentiation in hTS cells.

One embodiment provided herein describes the canonical Wnt pathway asinducing an inhibitory GSK3β, which results in the stabilization ofβ-catenin for nuclear translocation in cells. In another embodiment, RArapidly induces phosphorylation of GSK3β at Tyr216 site, downstreameffector of Akt2. In another embodiment, RA rapidly inducesphosphorylation of GSK3β at Tyr216 site, leading to the phosphorylationof β-catenin at the initial few hours that plays a ‘priming’ effect forthe later canonical Wnt pathway. In another embodiment, these activatedFzd6 and Dvl3 are able to facilitate the interaction of c-Jun N-terminalkinases (JNK) with the cytoskeleton or increase the intracellular Ca²⁺level, which in turn activates CaMKII for synaptic function in anon-canonical Wnt/Ca²⁺ signaling pathway. As time proceeds, a switchfrom non-canonical to canonical Wnt pathway occurs, attributing to thephosphorylation of GSK3β at Ser9/21 site. In one embodiment, G proteinregulates the transduction of non-canonical Wnt2B signaling at aninitial stage. In another embodiment, a canonical Wnt2B signaling occursat later stage in early developing neuronal differentiation.

HDAC6

Also provided herein is a method to induce hTS cells into neural stemcells, the method comprising modulating histone deacetylase 6 (HDAC6).Histone deacetylase 6 (HDAC6), an enzyme mainly located in thecytoplasm, regulates many biological processes, including cellmigration, immune synapse formation, viral infection, and thedegradation of misfolded proteins. For example, HDAC6 deacetylatestubulin, Hsp90 and cortactin, and forms complexes with other partnerproteins.

HDAC6 is capable of shuttling β-catenin for nuclear localization. In oneembodiment, HDAC6 interacts with β-catenin, leading to the nucleartranslocation of β-catenin by cellular fractionation assay. In anotherembodiment, RA induces a novel canonical Wnt2B/Fzd6/β-catenin signalingpathway, allowing nuclear translocation of β-catenin in hTS cells. Inthe nucleus, β-catenin involves in mediating key gene expressionprograms or as a docking platform for various transcriptionalco-activators to stimulate transcription.

HDAC4

Histone deacetylase 4 (HDAC4) is an important epigenetic regulator offunctional hTS cell-induced neural stem cells. HDAC4 inhibits cell-cycleprogression and protects neurons from cell death. Transcriptionalregulation by RARs involves modifications of chromatin by HDACs, whichare recruited to RA-target genes by nuclear co-repressors, determiningthe differential response to RA.

LEF/TCF/Pitx2

Lef-1 and PITX2 function in the Wnt signaling pathway by recruiting andinteracting with beta-catenin to activate target genes. PITX2 interactswith two sites within the Lef-1 protein. Furthermore, beta-catenininteracts with the PITX2 homeodomain and Lef-1 interacts with the PITX2C-terminal tail. Lef-1 and beta-catenin interact simultaneously andindependently with PITX2 through two different sites to regulate PITX2transcriptional activity. These data support a role for PITX2 in cellproliferation, migration, and cell division through differential Lef-1isoform expression and interactions with Lef-1 and beta-catenin.

Netrin 1 (NTN1)

The molecular mechanism of NTN1 is considered as primarily involved inaxonal guidance and control of neuronal cell migration.

Activation of Wnt/PS1/PI3K/Akt Pathway and Inhibition of GSK3-Beta by RA

Increased Wnt signaling expands the stem cell pool and forces expressionof a stabilized β-catenin resulting in a large brain owing to increasednumbers of proliferative progenitors and a corresponding decrease indifferentiated neurons (Chem, A. et al., Science 297, 365-369, (2002)).β-catenin has a dual role, as a junctional protein and in canonical Wntsignaling, the phenotype could be due to increased Wnt signaling (whichis linked to NSC self-renewal) or to increased junctional stability.

PI3K/Akt Signaling

Described herein, in one embodiment, is a method of maintainingpluripotency of tNSCs, the method comprising modulating the PI3K/Aktsignaling pathway. The G-protein beta/gammaheterodimers also activatePhosphoinositide-3-kinase, regulatory subunit 5 (PI3K regclass IB(p101)) that leads to Phosphoinositide-3-kinase, catalytic,gammapolypeptide (PI3K cat class IB (p110-gamma))-mediated conversion ofphosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) tophosphatidylinositol3,4,5-triphosphate (PtdIns(3,4,5)P3) [3].PtdIns(3,4,5)P3 is a second messenger that directly binds to3-phosphoinositide dependent protein kinase-1 (PDK(PDPK1)) and V-aktmurine thymoma viral oncogene homolog 1 (AKT(PKB)). PDK(PDPK1)phosphorylates AKT(PKB) and activates AKT signaling[4].

PI3K/Akt signaling regulates self-renewal and differentiation capacityin the following stem cell systems. The derivation of pluripotentembryonic germ (EG) cells from primordial germ cells (PGC) is enhancedin PGC-specific Pten-deficient mice (Kimura T, et al., Development 130:1691-1700, (2003)).

Using conditional activation of Akt signaling, it is shown that in oneembodiment, PI3K/Akt signaling plays a role in the activation of restingstem cells. In another embodiment, PI3K/Akt signaling plays a role inthe proliferation of progenitors in adult epidermis.

In one embodiment, PI3K/Akt signaling promotes the self-renewal of stemcells, rather than the generation of committed progenitors in theseculture-adapted stem cells. In one embodiment, RA modulates activationof Akt3/mTOR signaling that elicits the subcellular mRNAs translationencoding proteins RXRα and RARβ in hTS cells. In one embodiment, RAinduces activation of Akt3/mTOR signaling that elicits the subcellularmRNAs translation encoding proteins RXRα and RARβ in hTS cells. Inanother embodiment, an inducing agent inhibits activation of Akt3/mTORsignaling. In another embodiment, the selective movement andinteractions of the RXRα/Gα_(q/11) and RARβ/Gβ signaling pathways areinitiated independently.

In another embodiment, RA regulates genetic program transcriptionalactivities for cell functions depending on a pleiotropic and cellularcontext-dependent manner; i.e., the output phenotype is a combination ofthe effects of AP-1 and/or beta-catenin-LEF/TCF inhibition and RAREactivation.

GSK3/3 Regulates Microtubule Assembly

hTS cells embrace the major GSK3β functions that initial activation ofGSK3β promotes neuronal differentiation and later inactivation promotesprogenitor proliferation in neurodevelopment. In resting cells, thebasal activity of GSK3 is generally relative high while exposure of thecells to guidance cues can reduce its specific activity by between30-70% in 10 min. GSK3β has a strong preference for its substrates thatare already phosphorylated; therefore, the precedent primed β-cateninbecomes a favorable one for the later inhibitory GSK3β in the canonicalWnt2B signaling.

In one embodiment, the rapidly spatiotemporal active GSK3βphosphorylates MAPT localizes in axonal growth core, leading to theactivation of tubulin heterodimer (FIGS. 21a and 21b ) that promotemicrotubule assembly, neuronal polarity, and axon outgrowth consistentwith the notion that activation of GSK3β is involved in the axonalmicrotubule assembly. Moreover, GSK3β is also able to regulatephosphorylation of CRMP-2, contributing to microtubule assembly, wherebyCRMP-2 preferentially binds to tubulin heterodimer which is apparentlydistinct from that of MAPT. A mutant of CRMP-2 inhibits axonal growthand branching in a dominant-negative manner.

Provided herein in one embodiment is a mechanistic basis to assist inthe explanation in vivo that GSK-3 signaling is an important mediator ofhomeostatic controls that regulate neural progenitors in developmentalbrain. In another embodiment, the initial local activation of PI3K/Aktpathway induces activation of GSK3β at Tyr216 in hTS cells. In oneembodiment, initial local activation of PI3K/Akt pathway is distinctfrom the inactivation of GSK3β induced by Ser9/21 phosphorylation inhippocampal neurons isolated from E18 rat embryo. In one embodiment,phosphorylation at different sites in GSK3β results in differentcellular fate, depending on the time factor. Phosphorylated GSK3βprevents the DNA binding of calcineurin-induced NFAT1 by promotingnuclear export. NFAT plays a central role in promoting genetranscription, including cytokine genes in T-cells during the immuneresponses. These facts explain, at least partly, why both hTS cells andtNSCs possess immune advantages that facilitate intracranialtransplantation in PD rats.

G Protein and Neuronal Plasticity

The high degree of autonomy in NSCs permits rapidly local responses toguidance cues by the selective localization and translation of subsetsof mRNAs during neurogenesis. Wherein mTOR typically upregulates proteinsynthesis via phosphorylating key regulators of mRNA translation andribosome synthesis in NSCs. In hTS cells, active Akt3/mTOR signalingtriggers mRNA translation to independently synthesize RXRα and RARβproteins that activate Gα_(q/1) and Gβ signaling pathways, respectively.Wherein, local CREB1 is activated and plays a role of inducible geneexpression that transiently targets TH gene for transcription to produceneurotransmitter dopamine. It has been shown that RA promotes RARαexpression in the dendritic RNA granules and activates local glutamatereceptor 1 (GluR1) synthesis, implicating a homeostatic synapticplasticity. Therefore, an activation of dopamine D1/D5 receptor, theupstream enhancer of CREB, can induce GluR1 insertion at synaptic sitein neurons.

Provided herein, in one embodiment, is a molecular model for the studyof RA signal-related plasticity.

Transcription Factors for Dopaminergic Neurogenesis

In one embodiment, interaction of β-catenin and CREB1 in the nucleusrepresents a mainstream in TH transcription. In one embodiment, theactive β-catenin binds to lymphoid enhancer factor 1/T cell factor 1(LEF1), leading to the switch of LEF1 from repressor to activator oftranscription. LEF1 then recruited and interacted with Pitx2, member ofa superfamily of bicoid-related factor. In one embodiment, LEF1 promotesPitx2 gene transcription. In another embodiment, LEF1 promotes Pitx3gene. In another embodiment, LEF1 promotes both Pitx3 and Pitx2 genetranscription. In one embodiment, β-catenin, Pitx2, and LEF1synergistically interact to regulate the LEF-1 promoter.

Furthermore, the transient nuclear active NFAT1 plays as transcriptionfactor to produce cytokines and TNF-α for immune responses. However,this action was unlikely to occur in the present case because thephosphorylated GSK3β enables to inhibit the DNA binding ofcalcineurin-induced NFAT1 in the nucleus and to promote nuclear export.Therefore, active cytoplasmic NFAT1 would interact and activatecytoplasmic transcription factor myocyte enhancer factor 2A (MEF2A)(FIGS. 22c and 22d ) because this action was able to be inhibited byNFAT1 siRNA (FIG. 22e ). Notably, the rapid inducible CREB1 entered thenucleus and transcribed MEF2A gene that produced MEF2A protein (FIG. 22f). MEF2A might function in multiple ways at gene transcription (FIG. 22g), including transcription itself via auto-regulation to produce moreMEF2A, transcription TH gene for dopaminergic specification,transcription SNCA gene for SNCA/MAPT/parkin complex formation, andinteraction with EP300 and Pitx2, which was inhibited by MEF2A siRNA(FIG. 22h ).

In one embodiment, active ER300 targets the HDAC6 gene and the TH gene.In one embodiment, active ER300 targets the HDAC6 gene. In anotherembodiment, active ER300 targets the TH gene. In one embodiment, activeER300 promotes the transcription of the HDAC6 gene and the TH gene. Inanother embodiment, active ER300 inhibits the transcription of the HDAC6gene and the TH gene. In another embodiment, the HDAC6 transportsβ-catenin for nuclear translocalization.

Provided herein, in one embodiment, is the characterization of anexecutive transcription complex that is formed and destined for TH genetranscription. For example, CREB1, EP300, and MEF2A are able to targetthe promoter of the TH gene while β-catenin, LEF1, and Pitx2 perform asco-activators of the enhancer during transcription processes. Providedherein, in one embodiment, are methods to understand how these genesmanipulate the balance between differentiation and proliferation indopaminergic NSCs that have implications for the evaluation of diseasemechanisms (e.g., PD).

Multifarious Faces of CaMKII

In developing NSCs, local calcium influx through either voltage-gatedcalcium channels or neurotransmitter receptors results in the activationof CaMKII, delivering several messages forwards. In one embodiment, thespatiotemporal CaMKII triggers the c-Src mRNA localization via activatedeIF4B to synthesize c-Src protein, resulting in the activation of Nanogfor self-renewal and proliferation in hTS cells forexcitation-transcription coupling. In another embodiment, CAMKIItriggers activation of local CREB1, leading to a retrograde traffickingto the nucleus to target gene MEF2A for transcription. MEF2A mediatescellular functions not only in neuronal differentiation andproliferation, but also in skeletal and cardiac muscle development. Inone embodiment, CaMKII activates MAPT mediating parkin protein and inturn, MAPT activates tubulin heterodimer for microtubule assembly (FIGS.22a and 22j ). These results suggest that early spatiotemporal CaMKIIsignal is sufficient for the activation of tubulin to promotemicrotubule assembly, neuronal migration, and neuronal polarization inearly developing NSCs that ensure proper connectivity with striataltargets in the brain.

L-type calcium channels regulate intracellular calcium for homeostasisin another way, are involved in excitation-neurogenesis in adult NSCs.An elevated potassium chloride (KCl) level leads to membranedepolarization, resulting in an influx of calcium through L-typevoltage-sensitive calcium channels which is sufficient to inducemitochondrial dysfunction via the crosstalk between ER and mitochondriain neurons. In one embodiment, RA modulates intracellular ER calciumassociated with L-type calcium channels.

CaMKII (calmodulin (CaM)-dependent protein kinase II), a downstreameffector of L-type Ca²⁺ channels, exhibits a lower affinity forCa²⁺/calmodulin in response to transient low-amplitude calcium spikes.In one embodiment, RA modulates a spatiotemporal activation of CaMKII.In another embodiment, RA induces a spatiotemporal activation of CaMKII.In another embodiment, RA inhibits a spatiotemporal activation ofCaMKII.

CaMKII directly phosphorylated and activated CREB1 by IP assay (FIG. 21c) compatible with the previous study that CaMKII encodes L-type calciumchannel activity locally to signal to nuclear CREB inexcitation-transcription coupling. Since axons contains a variety ofmRNA encoding specific protein synthesis locally, including CaMKII,calcineurin, and CREB1 in developing neurons, suggesting the extrinsicRA-triggered mRNA translational machinery happens to them because theywere able to be inhibited by eukaryotic initiation factor 4B (EIF4B)siRNA (FIG. 21d ). Therefore, this local CREB1 enables the retrogradetrafficking for specific transcriptional processes in the nucleusresponsible for the signal of distal axons. These results suggested arapidly inducible gene transcription upon the extracellular cues.

These results first explored that the Gα_(q/11) signal-derived CaMKIIexcitation was involved in the maintenance of self-renewal of tNSCs.Together, these results suggested the importance of axonal behaviors inearly neurogenesis. SNCA interacts with the phospholipid membranes andplays crucial roles in the pathogenesis of neurodegenerative disordersincluding PD and Alzheimer's disease.

Calcineurin/NFAT1 Signaling

In one embodiment, RA modulates the production of calcineurin. In oneembodiment, RA induces production of calcineurin. In another embodiment,ER calcium is linked to calcineurin/NFAT1 signaling, consistent withprevious studies. In another embodiment, RA induces a transientinteraction of NFAT1 and importin, a nucleocytoplasmic transporter,leading to the NFAT1 nuclear translocation by cell fractionation assay.This temporal effect of NFAT1 is thought to be one mechanism by whichcells distinguish between sustained and transient calcium signals. Inone embodiment, RA-induced calcineurin/NFAT1 signaling is involved inthe early neurogenesis.

Cellular Remodeling at Initial Neurogenesis

Provided herein, in one embodiment, is a method for inducing molecularprocesses during the transition of hTS cells towards tNSCs. In oneembodiment, the molecular processes are induced by RA. In oneembodiment, the molecular cascades are examined at two time points: 4 hr(early) and 24 hr (later). In one embodiment, the molecular events occurin two phases. In a specific embodiment, one phase includes thespatiotemporal responses in morphogenesis (e.g., FIG. 23; early phase;grey line). In another specific embodiment, one phase includes the genetranscription in cell differentiation and proliferation (e.g., FIG. 23;later phase; black line).

In one embodiment, the mechanisms in early neuronal morphogenesis arecharacterized. Once the stem cells sense the external guidance signal, avariety of specific subcellular mRNA localizations initiate rapidly inresponsiveness to make up specific proteins locally beyond the fartranscription processes in the nucleus. Through the protein-proteininteraction and ‘sensory experience’ these local proteins accumulate atthe subcellular regions to initiate growth cone formation in earlydeveloping NSCs. In accompany with the gene transcription the asymmetricdivision begins. For instance, the presence of β-catenin is visible atthe synaptic membrane after RA treatment for 5 min (FIG. 23g ) and thelocal activated CREB1 travels back to the nucleus to target gene MEF2Afor transcription.

In one embodiment, a series of molecular processes synergistically occurto regulate mitochondrial function, lipid metabolism of membrane, axonalgrowth, neuronal migration and plasticity, and microtubule assembly,including but not limited to RXRα, RARβ, β-catenin, Akt, CREB1, mTOR,CaMKII, calcineurin, c-Src, GSK3β, SNCA, and MAPT. In anotherembodiment, the transcription at TH gene by MEF2A, EP300, and CREB1represent an inducible gene expression, which induces chromatin loopingfrom chromosome territories, facilitating the later gene transcription.In another embodiment, components of RA-induced G protein signaling playa key role in neuronal morphogenesis and also an integral part inactivating transcription at TH gene.

Described herein is a balance between the differentiation and theproliferation to maintain in a steady-state of tNSCs in vitro. In oneembodiment, neural differentiation is controlled by modulating RA-signaltransduction. Manipulation of hTS cells is enabled more efficiently invitro before further applications in regenerative medicine or drugdiscovery through the understanding of these regulatory mechanisms.

tNSCs Possess Immune Privilege

One embodiment provided herein describes a method of treating aneurological disorder using at least one tNSC wherein the cell is immuneprivileged. In another embodiment the tNSC does not elicit an immuneresponse. In another embodiment the tNSC does not elicit an immuneresponse from a T cell, B cell, macrophage, microglia, NK cell, or mastcell. In another embodiment the tNSC inhibits an immune response. Inanother embodiment the tNSC has reduced immunogenicity. In anotherembodiment, the tNSC does not lead to tumor formation. In anotherembodiment, the tNSC is designed to be immune privileged. In anotherembodiment provided herein describes a method of treating a neurologicaldisorder using a population of tNSC cells wherein the cells are immuneprivileged. In another embodiment, the application of stem cells ortheir derivatives as cell therapies benefits from the understanding oftheir immunogenicity to assist in the determination of application ofimmunosuppression agents postimplantation.

Another aspect described herein is a method to examine and compare theexpression of immune-associated genes and markers among hTS cells, tNSCsand hES cells. In one embodiment, expression is examined by flowcytometric analysis.

Examples of immune-associated genes and markers among hTS cells, tNSCsand hES cells include but are not limited to HLA-ABC, HLA-DR, CD14,CD44, CD73, CD33, CD34, CD45, CD105, and CD133. In another embodiment,the expression of HLA-ABC in hTS cells and tNSCs is higher in tNSCscompared to that in hES cells. In one embodiment, negative expression ofHLA-DR is observed in all three stem cells (FIG. 2e ). In anotherembodiment, the expression of HLA-ABC in hTS cells (99.4%) and tNSCs(99.7%) was much higher in tNSCs compared to that in hES cells (12.9%)(FIG. 2e ). In another embodiment, no difference in CD14 and CD44expressions was seen among hTS cells, tNSCs and hES cells. In anotherembodiment, high levels of CD73 were expressed in hTS cells and tNSCscompared to the negative expression levels in hES cells (FIG. 2f ). Inone embodiment, the tNSCs possess characteristics of mesenchymal stemcells, which are favorable for the proliferation of glial cells.

In another embodiment, CD33, which contains immunoglobulin structure atextracellular portion and is a transmembrane receptor, is expressed inhTS and hES cells but not tNSCs (FIG. 2f ). In another embodiment, theabsence of CD33 in tNSCs is in favor of cell therapy because of itsassociation with immune defense. Accordingly, provided herein are tNSCshaving low levels of expression of CD33 and thereby having lowimmunogenicity.

In one embodiment, no differences in intensities are found among them inthe expression of mesenchymal stem cell marker CD105. In anotherembodiment, low levels of expression of the cancer stem cell markerCD133 are found in tNSCs compared to hTS cells and hES cells. In anotherembodiment, low levels of expression of the cancer stem cell markerCD133 (11.8%) are found in tNSCs compared to hTS cells (93.6%) and hEScells (98.8%) (FIG. 2h ). Accordingly, provided herein are tNSCs havinglow levels of expression of CD133 and thereby having low tumorigenicity.

Further provided herein are selective populations of CD133+ tNSCs thatare useful for transplantation and tissue regeneration for stem celltherapy. Also provided herein are tNSCs with immune-privileged status,which are viable candidates for cell-based therapy.

In one embodiment, RA induces the changes in expression ofimmune-related markers, for example, cells with CD34(+) increased butwith CD133(+) decreased. In another embodiment, RA induces thedifferentiation of CD34(+) hES cells into smooth muscle progenitorcells. In another embodiment, autologous transplantation of tNSCs withCD34(+) immunoselected grafts is feasible in children with high-riskneuroblastoma.

Post-Implantation Differentiation and Proliferation

The association between RA and the retinoic acid-response element (RARE)in neurogenesis (Maden, M. et al., Nat. Rev. Neuroscience. 8, 755-765,(2007)) is known, however the existence of non-RARE action is poorlyunderstood. In one embodiment, RA induces activation of RXRα/RARβ/c-Srccomplex via “pull and push” mechanism of G protein-coupled receptors(GPCRs) signaling. In another embodiment, RXRα is first activated byinteraction with Gα_(q/11) followed by activation of c-Src and laterRARβ in 2 h to form a complex (FIGS. 3a and 3b ). Among them, c-Srcsubsequently induces Nanog overexpression through Stat3 for themaintenance of multipotency and self-renewal of those hTS cell-derivedNSCs.

This signaling pathway implies that it is not necessary for RA to enterthe cell to trigger the classical RA/RXR/RAR/RARE pathway, instead, RAactivates G protein Gα_(q/11) via GPCR signaling compatible with thenotion of signal transduction. Accordingly, provided herein, in oneembodiment, are methods for control of RA-mediated regulation ofmultipotency and self-renewal of NSCs, and manipulation of hTS cellsand/or neural stem cells before and after transplantation. In anotherembodiment, Wnt and RA impact caudal type homeobox 1 (Cdx1) through anatypical RARE and Lef/transcription factor (Tcf)-response elements(LRE), respectively, in the proximal promoter.

In one embodiment, RA induces hTS cells differentiation intodopaminergic NSCs via a classical RA/RARE signaling pathway to maintainthe stem cell properties. In another embodiment, is a non-RARE signalingpathway via activation of Wnt/β-catenin signaling cascade that generatesthe functional dopaminergic NSCs. In another embodiment, impairment ofthe non-RARE signaling causes dysfunction or loss of dopamineproduction, resulting in the progressive degenerative change ofdopaminergic neurons. Accordingly, provided herein, in anotherembodiment, is a neural stem cell that differentiates to dopaminergicneurons via activation of non-RARE signaling pathways.

RA activates the protein kinase C (PKC) pathway prior to induction ofRAR-β expression at 6 h. RA causes a transient 1.3-fold increase inintracellular diacylglycerol (DG) at 2 min and a translocation of thegamma isozyme of PKC (PKC-γ) within 5 min. Kurie J. M. et al., BiochimBiophys Acta. 1993, 1179(2):203-7. These findings reveal that PKCpathway activation is an early step in RA-mediated human TCdifferentiation and that PKC-γ can potentiate the effects of RA on RARtranscriptional activation. Accordingly, provided herein are methods tocontrol hTS cell differentiation. In one embodiment, modulation of thePKC signaling pathway controls hTS cell differentiation.

Bone morphogenetic protein 4 (BMP4) together with LIF supports expansionof undifferentiated mES cells. BMP4 induces trophoblasticdifferentiation of hES cells Qi X, et al., Proc Natl Acad Sci USA. 2004;101:6027-6032. BMP induction of Id proteins suppresses differentiationand sustains embryonic stem cell self-renewal in collaboration withSTAT3. Ying, Q. L., et al., Cell. 2003; 115:281-292. Bone morphogeneticproteins (BMPs) act in combination with LIF to sustain self-renewal andpreserve multilineage differentiation, chimera colonization, andgermline transmission properties. Xu R H, et al., Nat Biotechnol. 2002;20: 1261-1264. Accordingly, provided herein, in one embodiment, is amethod for inducing dopaminergic differentiation of tNSCs describedherein by modulation of PKC and/or Bone morphogenetic protein (BMP).

Treatment of Disease

Provided herein is a method to treat a disorder, wherein the methodcomprises transplanting a pure population of neurons or a complex ofspecific neural stem cell populations to a patient, wherein the patientis in need thereof. In one embodiment, the patient is diagnosed with aneurological disease. In another embodiment, the patient is diagnosedwith a neuropsychiatric disorder. In another embodiment, the patient isdiagnosed with a neurodegenerative disorder. In another embodiment, thepure population of neurons comprises dopaminergic neurons.

Any method described herein can be used to treat a disease or disorder.In one embodiment, the disease is a neurological disease. In anotherembodiment, the disease is a neurodegenerative disease or disorder.Non-limiting examples of neurological disorders include Parkinson'sdisease, Alzheimer's disease, Huntington's disease, Amyotrophic lateralsclerosis, Friedreich's ataxia, Lewy body disease, spinal muscularatrophy, multiple system atrophy, dementia, schizophrenia, paralysis,multiple sclerosis, spinal cord injuries, brain injuries (e.g., stroke),cranial nerve disorders, peripheral sensory neuropathies, epilepsy,prion disorders, Creutzfeldt-Jakob disease, Alper's disease,cerebellar/spinocerebellar degeneration, Batten disease, corticobasaldegeneration, Bell's palsy, Guillain-Barre Syndrome, Pick's disease, andautism.

Accordingly the tNSCs described herein are suitable for treatment ofneurodegenerative disorders including, and not limited to, Parkinson'sdisease, Alzheimer's disease, Huntington's disease, spinal cord injury,glaucoma, or the like.

In addition, the tNSCs also express neurotransmitter serotonin.Accordingly, one embodiment describes the use of tNSCs in treatment ofneuropsychiatric disorders. Non-limiting examples of neuropsychiatricdisorders include depression, schizophrenia, dementia, autism, attentiondeficit hyperactivity disorder, and dipolar disorder.

Any method described herein can be used to ameliorate or improve asymptom of a neurological disease or disorder. Non-limiting examples ofsymptoms associated with neurological disease or disorder includetremor, gait disorder, maldispositional gait, dementia, excessiveswelling (edema), muscle weakness, atrophy in the lower extremity,movement disorder (chorea), muscle rigidity, a slowing of physicalmovement (bradykinesia), loss of physical movement (akinesia),forgetfulness, cognitive (intellectual) impairment, loss of recognition(agnosia), impaired functions such as decision-making and planning,hemifacial paralysis, sensory deficits, numbness, tingling, painfulparesthesias in the extremities, weakness, cranial nerve palsies,difficulty with speech, eye movements, visual field defects, blindness,hemorrhage, exudates, proximal muscle wasting, dyskinesia, abnormalityof tonus in limb muscles, decrease in myotony, incoordination, wrongindication in finger-finger test or finger-nose test, dysmetria,Holmes-Stewart phenomenon, incomplete or complete systemic paralysis,optic neuritis, multiple vision, ocular motor disturbance such asnystagmus, spastic paralysis, painful tonic seizure, Lhermitte syndrome,ataxia, mogilalia, vesicorectal disturbance, orthostatic hypotension,decrease in motor function, bed wetting, poor verbalization, poor sleeppatterns, sleep disturbance, appetite disturbance, change in weight,psychomotor agitation or retardation, decreased energy, feelings ofworthlessness or excessive or inappropriate guilt, difficulty thinkingor concentrating, recurrent thoughts of death or suicidal ideation orattempts, fearfulness, anxiety, irritability, brooding or obsessiverumination, excessive concern with physical health, panic attacks, andphobias.

Described herein are tNSCs having certain desirable characteristics;first, the tNSCs are mixed cell populations composed of heterogeneoussubtypes with uniformity in phenotypes, stable gene expression andpluripotent characteristics; second, they contain glia progenitor cellsand astrocytes which substantially potentiate dopaminergic neurogenesis;third, they possess an intrinsic capacity to ‘rescue’ dysfunctionaldopaminergic neurons and the immune-privileged property; and finally,the neurotrophic effects secreted from different neural precursors onthe host tissue would facilitate structural repair.

Provided herein, in some embodiments, are tNSCs having certain desirablecharacteristics that allow for appropriate manipulation intransplantation therapy: 1) the unique tNSCs are simply and efficientlyinduced by RA in respect to consistency in quality and abundant cellsources; 2) the grafted tNSCs generate newly dopaminergic neurons in thelesioned nigrostriatal pathway functionally, which can survive for atleast 18 weeks postimplantation, 3) the sensorimotor impairments aresignificantly improved as early as from 3 weeks postimplantation; 4) thetNSCs possess immune privilege, facilitating stem cell therapy; 5)manipulations of the molecular mechanisms in cell proliferation asdescribed herein allows for development of strategies to preventtumorigenesis after transplantation; 6) the tNSCs are capable of beinggrown in culture through several cell passages; and 7) the tNSCs arecapable of being cultured in media that are free of mouse embryonicfeeder cells.

Provided herein, in one embodiment, is a method to treat acute andchronic disease, wherein the method comprises implantation of hTScell-derived tNSCs. In one embodiment, the tNSCs are implanted into thebrain of a patient suffering from a neurological disorder. In anotherembodiment, the tNSCs are implanted into the striatum of a patientsuffering from a neurological disorder.

One aspect described herein a method of treating a neurological disease,wherein the method comprises site-specific integration of tNSCs. In oneembodiment, the tNSCs are derived from hTS cells. In another embodiment,the chance of tumor formation is lower as compared to hES cell therapy.

Treatment of Neurodegenerative Diseases by Regeneration of DopaminergicNeurons

Provided herein are methods for inducing dopaminergic neurons in amammal wherein neuronal progenitor cells described herein aretransplanted as a cell suspension thereby producing a more homogenousreinnervation compared to transplants of tissue chunks. In oneembodiment, the induction of dopaminergic neurons as described hereinreduces the risk of dyskinesias and increases the chances of clinicallybeneficial effects. In one embodiment, the mammal is a human. In anotherembodiment, the mammal is a rat, mouse, pig, dog, monkey, orangutan orape.

Transplantation of tNSCs induces newly generated dopaminergic neurons inthe nigrostriatal pathway and substantially improved the behavioralimpairments in parkinsonian rats. These results provide evidence thathTS cells are human pluripotent stem cells that are suitable for use inclinical applications to treat neurodegenerative diseases.

A first experiment was conducted to examine: 1) whether the tNSCstreated with different duration of RA would affect the efficacy inimprovement of the behavioral deficits in PD rats and; 2) how long suchimplanted tNSCs can survive in the brain. Transplantation of theGFP-tagged tNSCs (1.5×10⁶) into two sites of the lesioned striatumsignificantly improved the behavioral defects from third week unto 12weeks by apomorphin-induced rotation assays (FIG. 5a ). PD rats received5-day RA-induced tNSCs improved significantly at the beginning of 6-weekpostimplantation, however, this effect lost henceforth similar to thatcontrol at 12 weeks. The reason can be explained by that most of theneurogenetically fate-restricted GRP (Götz) after induction over 5 daysare placed at a ridge in differentiating into undefined trophoblastgiant cells. Given the behavioral improvement, the rats were sacrificedat 18th week in order to examine the viability of those GFP-taggedtNSCs. Brain sections revealed abundant newly generated dopaminergicneurons in the nigrostriatal pathway with multiple outgrowths projectingfrom the cell body, reinnervating the surrounding brain areasimmunohistochemically (FIG. 5b ). However, no such phenomenon wasobserved in rats which received 5-day RA-induced tNSCs (FIG. 5c ) andthe control PD group (FIG. 5d ). Immunofluorescence microscopy, at 18thweek, demonstrated that the GFP-tagged tNSCs still existed in thelesioned areas, distributing in scattered or patchy patterns at theinjection site. Neither teratoma formation was found norimmunosuppression agent used.

To avoid the adverse effects from the dopaminergic overgrowth and unevenand patchy reinnervation, a second experiment was attempted totransplant less tNSCs (1×10⁶) by injection at one site into the lesionedstriatum in “aged” PD rats (n=16; body weight, 630-490 gm). Behavioralassessments were analyzed every 3 weeks postimplantation. Results showedthat there was a significant improvement of contralateral rotations from3-week toward 12-week postimplantation in the apomorphine-inducedrotation test (FIG. 6a ). To assess the effects of cell therapies in thepostural imbalance and gait disorder (PIGD), characterized by akinesia,rigidity and gait and balance impairments, several tests were performedsuch as walking speed, step length, stride length and base of support.The grasping time of the affected forelimb on the bar was significantlyshortened by 3 weeks and continued to improve at the end of 12 weeks inthe “bar test” (FIG. 6b ), indicating a very quick improvement in thepower of seizure in forelimb. Measurements of step length (FIG. 6c ),stride length (FIG. 6d ), walking speed (FIG. 6e ) and base of support(FIG. 6f ) showed that transplantation of the tNSCs significantlyimproved the sensorimotor impairments from early 3-week towards 12-weekfunctionally. In one embodiment, the tNSCs are suitable candidates forstem cell-based therapy in patients with neurodegenerative diseases(e.g., Parkinson's disease) in regenerative medicine. At the end of 12weeks, rats were sacrificed and brain sections were subjected fortyrosine hydroxylase (TH) immunostaining. The experiments showedregeneration of new dopaminergic neurons appeared in the nigrostriatalpathway (FIG. 19). The newly generated dopamine neurons were assessed byusing densitometry, which revealed a 28.2% in recovery. In oneembodiment, the tNSCs are an alternative substitute of both hES cellsand fetal mesencephalic tissue in the treatment of patients withneurodegenerative diseases.

Provided herein, in one embodiment, is a hTS cell that is a humanpluripotent stem cell other than a hES cell but with similarcharacteristics of pluripotency and self-renewal in early embryogenesis.In vivo, the grafted tNSCs generate newly dopaminergic neurons in thelesioned nigrostriatal pathway functionally, which can survive for atleast 18 weeks postimplantation in PD rats. Sensorimotor impairments aresignificantly improved as early as from 3 weeks postimplantation by aset of behavioral assessments in both young and aged PD rats.Transplantation of the hTS cell-derived NSCs into theneurotoxin-denervated striatum of brain enables regeneration of the lostdopaminergic neurons and improves the major behavioral deficits in ratswith PD.

In one embodiment, DA neurons in the nigrostriatal pathway areregenerated. In another embodiment, the implanted tNSCs increase glialcells in the striatum. In another embodiment, RA induces the expressionof GRAD and GFAP-positive progenitor cells, giving rise to neurons andoligodendrocytes throughout the CNS.

Treatment of Alzheimer's Disease

Provided herein are methods for treating Alzheimer's Disease, whereinthe method comprises transplanting neuronal progenitor cells into thebrain of a mammal. In one embodiment, the mammal is a human. In anotherembodiment, the human is a patient diagnosed with Alzheimer's Disease orat risk of developing Alzheimer's Disease, e.g., a person with a familyhistory of the disease or who has been identified as having a riskfactor for the disease. In another embodiment, the mammal is a pig, dog,monkey, orangutan or ape. In another embodiment, the mammal is a mouse.In another embodiment, the mammal is a rat. In another embodiment, therat or mouse displays symptoms of Alzheimer's Disease. In oneembodiment, the neuronal progenitor cells are transplanted into anon-human animal model for the disease (e.g., a mouse model in whichAD7c-NTP is overexpressed, an Alzheimer's Disease rat model, atransgenic mouse model, etc.)

In one embodiment, hTS cells are treated with an inducing agent toprovide a neuronal cell population with a biomarker signature. In aspecific embodiment, the inducing agent is RA. In one embodiment, themolecular mechanisms or signaling pathways are modulated to maintainpluripotency. In another embodiment, the molecular mechanisms orsignaling pathways are modulated to prevent tumorigenesis aftertransplantation.

In another embodiment, the tNSCs are grafted or inserted into the brainof the mammal. In one embodiment, the neuronal progenitor cells aretransplanted as a cell suspension thereby producing a more homogenousreinnervation. In another embodiment, the neuronal progenitor cells areinjected into the brain of said mammal. In another embodiment, the tNSCsderived from hTS cells are inserted into the subventricular zone of thebrain. In one embodiment the mammal is a human.

In one embodiment, the induction of neurons as described herein reducesthe risk of tumorigenesis and increases the chances of clinicallybeneficial effects. In another embodiment, the recipient of the tNSCsshows an improvement in symptoms associated with Alzheimer's disease. Inanother embodiment, the connections between neurons in the brain areincreased and strengthened.

Treatment of Schizophrenia

Provided herein are methods for treating schizophrenia, wherein themethod comprises transplanting neuronal progenitor cells into the brainof a mammal. In one embodiment, the mammal is a human. In anotherembodiment, the human is a patient diagnosed with schizophrenia or atrisk of developing schizophrenia, e.g., a person with a family historyof the disease or who has been identified as having a risk factor forthe disease. In another embodiment, the mammal is a mouse. In anotherembodiment, the mammal is a rat. In another embodiment, the mammal is apig, dog, monkey, orangutan or ape. In another embodiment, the rat ormouse displays symptoms of schizophrenia.

In one embodiment, the neuronal progenitor cells are transplanted into anon-human animal model for the disease (e.g., a schizophrenia rat model,a transgenic mouse model, etc.) In one embodiment, model mouse has analtered normal physiological regulation of the neuronal system. Inanother embodiment, the model animal or tissues can be utilized forscreening of potential therapeutic agents and/or therapeutic regimensthat act at the intracellular level.

In one embodiment, hTS cells are treated with an inducing agent toprovide a neuronal cell population with a biomarker signature. In aspecific embodiment, the inducing agent is RA. In one embodiment, themolecular mechanisms or signaling pathways are modulated to maintainpluripotency. In another embodiment, the molecular mechanisms orsignaling pathways are modulated to prevent tumorigenesis aftertransplantation.

In another embodiment, the tNSCs are grafted or inserted into the brainof the mammal. In one embodiment, the neuronal progenitor cells aretransplanted as a cell suspension thereby producing a more homogenousreinnervation. In another embodiment, the neuronal progenitor cells areinjected into the brain of said mammal.

In one embodiment, the induction of neurons as described herein reducesthe risk of tumorigenesis and increases the chances of clinicallybeneficial effects. In another embodiment, the recipient of the tNSCsshows an improvement in symptoms associated with schizophrenia.

Dosing and Administration

Modes of administration of an isolated neural stem cell preparationdescribed herein include, but are not limited to, systemic intravenousinjection and injection directly to the intended site of activity. Thepreparation can be administered by any convenient route, for example, byinfusion or bolus injection, and can be administered together with otherbiologically active agents. In one embodiment, administration issystemic localized administration.

In one embodiment, a neural stem cell preparation or composition isformulated as a pharmaceutical composition adapted for intravenousadministration to mammal, including human beings. In some embodiments,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition also includesa local anesthetic to ameliorate any pain at the site of the injection.Where the composition is to be administered by infusion, it can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the composition is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients are mixed prior to administration.

In one embodiment, suitable pharmaceutical compositions comprise atherapeutically effective amount of the progenitor stem cells and apharmaceutically acceptable carrier or excipient. Such a carrierincludes, but is not limited to, saline, buffered saline, dextrose,water, and combinations thereof.

In one embodiment, the isolated tNSCs described herein are delivered toa targeted site (e.g., the brain, the spinal cord or any other site ofnerve injury and/or degeneration) by a delivery system suitable fortargeting cells to a particular tissue. For example, the cells areencapsulated in a delivery vehicle that allows for the slow release ofthe cell(s) at the targeted site. The delivery vehicle is modified suchthat it is specifically targeted to a particular tissue. The surface ofthe targeted delivery system is modified in a variety of ways. In thecase of a liposomal-targeted delivery system, lipid groups areincorporated into the lipid bilayer of the liposome in order to maintainthe targeting ligand in stable association with the liposomal bilayer.

In another example, a colloidal dispersion system is used. Colloidaldispersion systems include macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems, including oil-in-wateremulsions, micelles, mixed micelles, and liposomes.

The administration of tNSCs described herein is optionally tailored toan individual, by: (1) increasing or decreasing the amount cellsinjected; (2) varying the number of injections; (3) varying the methodof delivery of the cells; or (4) varying the source of cells, e.g., bygenetically engineering cells, or from in vitro cell culture.

The tNSC preparation is used in an amount effective to promoteengraftment of cells in the recipient. At the physician's discretion,the administration is adjusted to meet optimal efficacy andpharmacological dosing.

Methods of Screening

Provided herein are methods of screening a compound for use in treatmentor prevention of a disease. In one embodiment, the method comprisescontacting an isolated human trophoblastic stem cell with said compound.In another embodiment, the method comprises contacting an isolatedneural stem cell with said compound. In another embodiment, the methodfurther comprises detecting a change in the activity of at least onegene, transcript or protein in said human trophoblastic stem cell. Inanother embodiment, the method further comprises detecting a change inthe level of at least one transcript or protein in said humantrophoblastic stem cell. In another embodiment, the method comprisesdetecting a change in the activity of at least one gene, transcript orprotein in said neural stem cell.

One embodiment provided herein describes a method of screening acompound for ability to induce changes in a cell comprising. In oneembodiment, the method comprises contacting an isolated humantrophoblastic stem cell with said compound. In another embodiment, themethod comprises contacting an isolated neural progenitor stem cell withsaid compound. In another embodiment, the method further comprisesdetecting an induction of differentiation of said human trophoblasticstem cell. In another embodiment, the method further comprises detectingan induction of differentiation of said neural stem cell.

Also provided herein a method of screening a compound for cellulartoxicity or modulation of the cell, the method comprising contacting adifferentiated cell of this invention with the compound. In anotherembodiment, the method further comprises determining any phenotypic ormetabolic changes in the cell that result from contact with thecompound, and correlating the change with cellular toxicity or any otherchange in cell function or biochemistry. In another embodiment,screening of pharmaceuticals, toxins, or potential modulators ofdifferentiation is facilitated. These substances (e.g., pharmaceuticals,toxins, or potential modulators) can be added to the culture medium.

One embodiment provided herein described a method of screeningproliferation factors, differentiation factors, and pharmaceuticals. Inone embodiment, human trophoblast stem cell or neural stem cell are usedto screen for factors (such as small molecule drugs, peptides,polynucleotides, and the like) or conditions (such as culture conditionsor manipulation) that affect the characteristics of human trophoblaststem cell or neural stem cell in culture. In one embodiment, this systemhas the advantage of not being complicated by a secondary effect causedby perturbation of the feeder cells by the test compound. In anotherembodiment, growth affecting substances are tested. In anotherembodiment, the conditioned medium is withdrawn from the culture and asimpler medium is substituted. In another embodiment, different wellsare then treated with different cocktails of soluble factors that arecandidates for replacing the components of the conditioned medium.Efficacy of each mixture is determined if the treated cells aremaintained and proliferate in a satisfactory manner, optimally as wellas in conditioned medium. Potential differentiation factors orconditions can be tested by treating the cell according to the testprotocol, and then determining whether the treated cell developsfunctional or phenotypic characteristics of a differentiated cell of aparticular lineage.

In one embodiment, the human trophoblast stem cell or neural stem cellare used to screen potential modulators of cellular differentiation. Inone embodiment, the cellular differentiation is neural differentiation.For example, in one assay for screening modulators of cellulardifferentiation, the human trophoblast stem cell or neural stem cell canbe cultured under serum free, low density conditions in the presence orabsence of LIF, in the present of the modulator, and in the present orabsence of RA, as the situation requires, and the effect ondifferentiation can be detected. In another embodiment, the screeningmethods described herein can be used to study conditions associated withcellular development and screen for potential therapeutic or correctivedrugs or modulators of the condition. For example, in one embodiment,the development of the normal human trophoblast stem cell or neural stemcell is compared with the development with cells having the condition.

In one embodiment, gene and protein expression can be compared betweendifferent cell populations obtained from human trophoblast stem cell orneural stem cell, and used to identify and characterize factorsupregulated or downregulated in the course of differentiation, andproduce nucleotide copies of the affected genes.

In one embodiment, feeder-free human trophoblast stem cell or neuralstem cell cultures can also be used for the testing of pharmaceuticalcompounds in drug research. Assessment of the activity of candidatepharmaceutical compounds generally involves combining the differentiatedcells of this invention with the candidate compound, determining anyresulting change, and then correlating the effect of the compound withthe observed change. In another embodiment, the screening is done, forexample, either because the compound is designed to have apharmacological effect on certain cell types, or because a compounddesigned to have effects elsewhere have unintended side effects. Inanother embodiment, two or more drugs are be tested in combination (bycombining with the cells either simultaneously or sequentially), todetect possible drug-drug interaction effects. In another embodiment,compounds are screened initially for potential toxicity. In anotherembodiment, cytotoxicity is be determined by the effect on cellviability, survival, morphology, on the expression or release of certainmarkers, receptors or enzymes, on DNA synthesis or repair.

The terms “treating,” “treatment,” and the like are used herein to meanobtaining a desired pharmacologic and/or physiologic effect. In someembodiments, an individual (e.g., an individual suspected to besuffering from and/or genetically pre-disposed to a neurodegenerativedisorder is treated prophylactically with a preparation of tNSCsdescribed herein and such prophylactic treatment completely or partiallyprevents a neurodegenerative disorder or sign or symptom thereof. Insome embodiments, an individual is treated therapeutically (e.g., whenan is suffering from a neurodegenerative disorder), such therapeutictreatment causes a partial or complete cure for a disorder and/orreverses an adverse effect attributable to the disorder and/orstabilizes the disorder and/or delays progression of the disorder and/orcauses regression of the disorder.

Administration (e.g., transplantation) of tNSCs to the area in need oftreatment is achieved by, for example and not by way of limitation,local infusion during surgery, by injection, by means of a catheter, orby means of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers.

“Transplanting” a composition into a mammal refers to introducing thecomposition into the body of the mammal by any method established in theart. The composition being introduced is the “transplant”, and themammal is the “recipient”. The transplant and the recipient can besyngeneic, allogeneic or xenogeneic. Further, the transplantation can bean autologous transplantation.

An “effective amount” is an amount of a therapeutic agent sufficient toachieve the intended purpose. For example, an effective amount of afactor to increase the number of hTS cells or tNSCs is an amountsufficient, in vivo or in vitro, as the case can be, to result in anincrease in neural stem cell number. An effective amount of acomposition to treat or ameliorate a neurodegenerative disease orcondition is an amount of the composition sufficient to reduce or removethe symptoms of the neurodegenerative disease or condition. Theeffective amount of a given therapeutic agent will vary with factorssuch as the nature of the agent, the route of administration, the sizeand species of the animal to receive the therapeutic agent, and thepurpose of the administration.

Further provided herein in one embodiment are genetically modifiedtNSCs. Manipulations modify various properties of the cell, e.g., renderit more adapted or resistant to certain environmental conditions, and/orinduce a production of one or more certain substances therefrom, whichsubstances can, e.g., improve the viability of the cell. Such geneticalterations can be performed in order to make the cell more suitable foruse in transplantation, for example, in order to avoid rejection thereoffrom the recipient (for reviews of gene therapy procedures, seeAnderson, Science, 256:808; Mulligan, Science, 926; Miller, Nature,357:455; Van Brunt, Biotechnology, 6(10):1149; and Yu et al., GeneTherapy, 1:13).

A “vector” refers to a recombinant DNA or RNA construct, such as aplasmid, a phage, recombinant virus, or other vector that, uponintroduction into an appropriate host cell, results in a modification ofa progenitor cell described herein. Appropriate expression vectors arewell known to those with ordinary skill in the art and include thosethat are replicable in eukaryotic and/or prokaryotic cells and thosethat remain episomal or those that integrate into the host cell genome.

Construction of vectors is achieved using techniques described in, forexample, as described in Sambrook et al., 1989. In one embodimentisolated plasmids or DNA fragments are cleaved, tailored, and religatedin the form desired to generate the plasmids. If desired, analysis toconfirm correct sequences in the constructed plasmids is performed usingany suitable method. Suitable methods for constructing expressionvectors, preparing in vitro transcripts, introducing DNA into hostcells, and performing analyses for assessing gene expression andfunction are known. Gene presence, amplification, and/or expression aremeasured in a sample directly, for example, by conventional Southernblotting, Northern blotting to quantitate the transcription of mRNA, dotblotting (DNA or RNA analysis), or in situ hybridization, using anappropriately labeled probe which can be based on a sequence providedherein.

As used herein, terms such as “transfection”, “transformation”, and thelike are intended to indicate the transfer of nucleic acid to a cell ororganism in functional form. Such terms include various means oftransferring nucleic acids to cells, including transfection with CaP04,electroporation, viral transduction, lipofection, delivery usingliposomes, and/or other delivery vehicles.

Cells are sorted by affinity techniques or by cell sorting (such asfluorescence-activated cell sorting) where they are labeled with asuitable label, such as a fluorophore conjugated to or part of, forexample, an antisense nucleic acid molecule or an immunoglobulin, or anintrinsically fluorescent protein, such as green fluorescent protein(GFP) or variants thereof. As used herein, “sorting” refers to the atleast partial physical separation of a first cell type from a second.

As used herein, the term “about” means±15%. For example, the term “about10” includes 8.5 to 11.5.

EXAMPLES Materials

Antibodies.

For immunoblot and immunocytochemistry: primary antibodies: SSEA-1, -2,-3, CD90 and nestin (Chemicon). Neurofilament, and GFAP (BioGenex).Nanog, Oct4, Cdx2 and Sox2 (BD Biosciences, San Jose, Calif., USA).G_(αq/11) (C-19, sc-392), Gβ (T-20, sc-378), RXRα, RARβ, c-Src, pStat3,Stat3, PP1 analog and β-actin (Santa Cruz Biotechnology, Santa Cruz,Calif., USA), TH (Sigma-Aldrich St. Louis, Mo. and Temcoula, Calif.) andserotonin (Sigma-Aldrich St. Louis, Mo.).

Secondary Antibodies:

siRNAs: Nanog siRNA and Cdx2 siRNA (Sigma-Aldrich St. Louis, Mo.).

For flow cytometry Primary antibodies: HLA-ABC, CD9, CD14, CD34, CD45,CD73, CD90, CK7, vimentin, 6-integrin, E-cadherin, L-selectin, Nanog,Oct4, Cdx2 and Sox2 were purchased from BD Biosciences, San Jose,Calif., USA; HLA-DR, CD33, CD44 and CD105 from eBioscience, San Diego,Calif., USA; CD133 from Miltenyi Biotec, Germany.

For TH-2 and serotonin immunostainings, cells were incubated in 0.1M PBSat 4° C. overnight after washing with PBS. After incubation withblocking solution (50 ml 0.1 M PBS, 0.05 g sodium azide, 1% horse serumand 10% Triton X-100) for 1 h at room temperature, the cells were washedagain. Cells were incubated with primary antibody, i.e., TH-2 (1:200,Sigma-Aldrich, St. Louis, Mo.) and serotonin (1:100, Sigma-Aldrich, St.Louis, Mo.) for 2 h and washed with PBS. By incubation with anti-mouseIgG with FITC or PE (Sigma-Aldrich, St. Louis, Mo.) for 1 h, cells werethoroughly washed with PBS and subjected to immunofluorescence assays.

Example 1 Isolation, Differentiation and Cell Culture

Embryonic chorionic villious were obtained from the fallopian tubes ofearly ectopic pregnancy (gestational age: 6-8 weeks) in women vialaparoscopic surgery, approved by the Institutional Review Board onHuman Subjects Research and Ethics Committees. Tissues were minced inserum-free α-MEM (Sigma-Aldrich, St. Louis, Mo.) and trypsinized with0.025% trypsin/EDTA (Sigma-Aldrich, St. Louis, Mo.) for 15 min and thisdigestion was halted by adding α-MEM containing 10% FBS. This procedurewas repeated several times. After centrifugation, cells were collectedand cultured with α-MEM containing 20% FBS (JRH, Biosciences, San Jose,Calif.) and 1% penicillin-streptomycin in 5% CO₂ at 37° C. The hCGexpression in the medium became undetectable after two passages ofculture measured by a commercial kit (Dako, Carpinteria, Calif.).

Cell Differentiation.

hTS cells were cultured in conditioned α-MEM containing 20% FBS, 1%penicillin-streptomycin, and 10 μg/ml bFGF (CytoLab Ltd, Rehovot,Israel) at 37° C. in 5% CO₂. The medium was replaced every 3 days. Afterfive passages, differentiation into various specialized phenotypes wasinitiated by using published protocols with modifications. For cellculture in Transwell plate (Corning, New York, N.Y.), the upper chamberwas coated with 500 μl of collagen gel containing PureCol (InamedBiomaterials, Fremont, Calif.) and conditioned L-DMEM (Gibco, GrandIsland, N.Y.) at a 4:1 ratio (adjusted to pH 7.4 using 1 M NaHCO₃).Cells (4×10⁵) were cultured in conditioned L-DMEM (1 ml) on the upperchamber. The lower chamber contained conditioned H-DMEM (3 ml).Preliminary experiments showed that the glucose levels in both chamberscould reach an equilibrium status in 4 hr.

Cell Differentiation of Subphenotypes.

Cells were cultured in conditioned α-MEM containing 20% FBS, 1%penicillin-streptomycin, and 10 μg/ml bFGF (CytoLab Ltd, Rehovot,Israel) at 37° C. in 5% CO₂. The medium was refreshed every 3 days ingeneral. After 5 passages of culture, cell differentiations into avariety of specific cell phenotypes were performed by various strategiesas shown in the Table in FIG. 12. For osteogenic differentiation,cytochemical mineral matrix was analyzed using an Alizarin red S assay(Sigma-Aldrich, St. Louis, Mo.) to detect the calcium mineral content.To identify the calcium deposits, cells were fixed and incubated with 2%silver nitrate solution (w/v) for 10 min in dark followed by thoroughwash with de-ionized water and exposed under bright light for 15 min.Cells were treated with von Kossa staining to detect alkalinephosphatase activity using a commercial kit (Sigma-Aldrich, St. Louis,Mo.). Chondrogenic differentiation was confirmed using Alcian bluestaining (Sigma-Aldrich, St. Louis, Mo.) at an acidic pH level. Formyogenic differentiation, cells were incubated with 3% hydrogen peroxidein phosphate buffer saline (PBS) for 10 min to quench the endogenousperoxidase enzyme activity. The non-specific sites were blocked by PBScontaining 10% human serum and 0.1% Triton X-100 for 60 min and washedby blocking buffer for 5 min. Cells were incubated in blocking buffercontaining skeletal muscle myosin heavy chain-specific monoclonalantibody (Vector Laboratories, Burlingame, Calif.) for 1 h, and stainedusing VectaStain ABC kit (Vector Laboratories). For adipogenicdifferentiation, cells were induced by conditioned media and fixed for60 min in 4% paraformaldehyde containing 1% calcium and washed with 70%ethanol. After exposure to 2% Oil red 0 reagent (Sigma-Aldrich, St.Louis, Mo.) for 5 min, the excessive staining was removed by 70% ethanolfollowed by water rinses. Oil red O stain was applied as an indicator ofintracellular lipid accumulation. Neural stem cells were induced by 10μM all-trans retinoic acid (Sigma-Aldrich, St. Louis, Mo.) in ethanol.

Example 2 Plasmid Transfection

For plasmid transfection, hTS cells were induced by all-trans retinoicacid (10 μM) (Sigma-Aldrich, St. Louis, Mo.) overnight followed byco-transfection in a DNA mixture of F1B-GFP as described previously(Myers). Briefly, the DNA mixture was added slowly into DOTAP (100 μl)solution containing DOTAP (30 μl) liposomal transfection reagent (RocheApplied Science, Indianapolis, Ind.) and 70 μl HBSS buffer containingNaCl (867 g in 80 ml H₂O) plus 2 ml HEPES solution (1 M, pH 7.4, Gibco)at 4° C. for 15 min. After wash by PBS, cells were mixed well with theDNA mixture. After incubation overnight, stable cells lines wereobtained by G418 selection (400 μg/ml, Roche Applied Science) throughculture for 2-3 weeks until the formation of colonies. TheG418-resistant cells were pooled and lysed and analyzed by Westernblotting using monoclonal anti-GFP antibody (Stratagene, La Jolla,Calif.) to quantify the percentage of transfectants that expressed GFP.By subcultures, the transfected hTS cells were fixed with methanol (10min) to detect the expression of GFP by immunofluorescence. Thetransfection rate yielded over 95% of efficacy.

Example 3 RT-PCR and Quantitative PCR (qPCR)

For RT-PCR, total RNA from 10⁵-10⁶ cells was extracted by using TRIZOLreagent (Invitrogen) and mRNA expression by using a Ready-To-Go RT-PCRBeads kit (Amersham Biosciences, Buckinghamshire, UK). Briefly, thereaction products were resolved on 1.5% agarose gel and visualized withethidium bromide. β-actin or β-2 microglobulin was used as a positivecontrol. All experiments were performed in triplicate. For qPCR, geneexpression was measured with the iQ5 Real-Time PCR Detection System(Bio-Rad Laboratories) and analyzed with Bio-Rad iQ5 Optical SystemSoftware, version 2.0 (Bio-Rad Laboratories). Relative mRNA levels werecalculated using the comparative Ct method (Bio-Rad, instruction manual)and presented as a ratio to biological controls. All primer pairs wereconfirmed to approximately double the amount of product within one cycleand to yield a single product of the predicted size.

Example 4 Western Blots

Cells were seeded into 10 cm dish with the serum-free medium forovernight and treated with or without RA (10 μM) for various timeintervals as indicated. After stimulation, cells were washed twice withice-cold PBS and lysed by RIPA lysis buffer (Minipore). Proteinconcentration was determined by BCA protein assay kit (Thermo). Equalamounts of protein (30 μg) were resolved by 8% SDS-PAGE, transferredonto PVDF membrane and blocked with 5% non-fat dry milk for 1 h at roomtemperature. After blocking, the membrane was incubated with primaryantibodies for 4 h at 4° C. Cells were washed three times with PBST andthen incubated with HRP conjugated secondary antibodies for 1 h at roomtemperature. After washing six times with PBST buffer, the membrane wasincubated with a chemiluminescent substrate (GE Healthcare) for 1 min.Specific bands were visualized using an enhanced chemiluminescence kit(ECL) (Amersham).

Example 5 Southern Blots

The telomere length of hTS cell was measured at passages 3 and 7 bysouthern immunoblot analysis as described previously (Tsai). Briefly,the fragments were transferred to Hybond N+ nylon membranes (AmershamBiosciences) and hybridized at 65° C. to a probe of TTAGGG repeatslabeled with α-³²P-dCTP using Ready-To-Go labeling beads (AmershamBiosciences). Terminal restriction fragments were visualized byhybridization with labeled oligonucleotides complementary to thetelomeric repeat sequence. The size distribution of the TRFs wascompared with a DNA length standard.

Example 6 Terminal Restriction Fragment (TRF) Southern Blot

Since a cell initiates its cancerous change, its telomeres would becomevery short. The tolomere length was measured at 3^(rd) and 7^(th)passages in culture of hTS cells. Briefly, the fragments weretransferred to Hybond-N+ nylon membranes (Amersham Biosciences) andhybridized at 65° C. to a probe of TTAGGG repeats labeled withα-[³²P]-dCTP by using Ready-To-Go DNA Labeling Beads (AmershamBiosciences). Terminal restriction fragments were visualized byhybridization with labeled oligonucleotides complementary to thetelomeric repeat sequence. The size distribution of the terminalrestriction fragments was compared with a DNA length standard. Forelectron microscopy, the hTS cell-derived grape-like cell mass wasexamined by transmission electron microscopy (JEM-2000 EXII, JEOL,Tokyo, Japan) to identify the infrastructure of the cell.

The differential gene expressions of Oct4, Sox2, NANOG, fgfr2, FGF4,BMP4, Cdx2, and endogenous control β-actin (ACTB) were measured in thehTS and the hTS cells treated by 500 units LIF (Chemicon, Temecula,Calif.) by IQ5 Real-time PCR detection system (Bio-Rad Laboratories)that using fluorescein as an internal passive reference dye fornormalization of well-to-well optical variation. PCR amplifications werecarried out in a total volume of 25 μl, containing 12.5 μl of 2×SYBRGreen supermix (Bio-Rad), 0.5 μl of 10 μM of each primer and 0.5 μl ofcDNA samples and mixed with sterile water. The reaction was initiated at95° C., 3 minutes, followed by 60 three-step amplification cyclesconsisting of 30 s denaturation at 95° C., 30 s annealing at 60° C., 15s extension at 72° C. At final dissociation stage, it was run togenerate a melting curve for verification of amplification productspecificity. Real-time qPCR was monitored and analyzed by the Bio-RadIQ5 optical system software version 2.0 (Bio-Rad). Relative mRNA levelswere calculated using the comparative Ct method (Bio-Rad instructionmanual) and presented with ratio to biological controls. ACTB transcriptlevels were confirmed to correlate well with total RNA amounts andtherefore used for normalization throughout. All primer pairs used wereconfirmed to approximately double the amount of product within one cycleand to yield a single product of the predicted size. Primer sequences ofOct4, Sox2, NANOG, fgfr2, FGF4, BMP4, Cdx2, and endogenous controlβ-actin (ACTB) are shown in Supplemental Data Table 3.

(SEQ ID NO: 1) OCT4-F: CCATCTGCCGCTTTGAGG; (SEQ ID NO: 2) OCT4-R:ACGAGGGTTTCTGCTTTGC; (SEQ ID NO: 3) ACTB-F: GATCGGCGGCTCCATCCTG;(SEQ ID NO: 4) ACTB-R: GACTCGTCATACTCCTGCTTGC; (SEQ ID NO: 5) CDX2-F;GTGTACACGGACCACCAGCG (SEQ ID NO: 6) CDX2-R; GGTGGCTGCTGCTGCTGTTG(SEQ ID NO: 7) MIG7-F; TCCACTACCAAGAGACAGGCTT (SEQ ID NO: 8) MIG7-R;TCAAGCTGTGTTGCACCCAA (SEQ ID NO: 9) IPF-1-F; GGAGGAGAACAAGCGGACGC(SEQ ID NO: 10) IPF-1-R; CGCGCTTCTTGTCCTCCTCC

TABLE 1 Various PCR primers (SEQ ID NOS 11-48, respectively,in order of appearance) used for gene expression Product Anneal GeneSequence (5′→3′) size (bp) temp ° C. OsteopontinForward:: CTAGGCATCACCTGTGCCATACC 330 55.7 Reverse:CAGTGACCAGTTCATCAGATTCATC Osteocalcin Forward: CGCAGCCACCGAGACACCAT 40566 Reverse: GGGCAAGGGCAAGGGGAAGA Perlecan Forward: CATAGAGACCGTCACAGCAAG300 50 (PRLN) Reverse: ATGAACACCACACTGACAACC CollagenForward: ACGGCGAGAAGGGAGAAGTTG 352 60.1 typeIIReverse: GGGGGTCCAGGGTTGCCATTG Myogenin Forward: AGCGCCCCCTCGTGTATG 36561 Reverse: TGTCCCCGGCAACTTCAGC MyoD1 Forward: CGGCGGCGGAACTGCTACGAA 45265.8 Reverse: GGGGCGGGGGCGGAAACTT PPARγ-2 Forward: GCTGTTATGGGTGAAACTCTG352 50.7 Reverse: ATAAGGTGGAGATGCAGGCTC AdipsinForward: GGTCACCCAAGCAACAAAGT 269 61 Reverse: CCTCCTGCGTTCAAGTCATC β2-Forward: 335 57.3 microglobulin CTCGCGCTACTCTCTCTCTTTCTGG Reverse:GCTTACATGTCTCGATCCCACTTAA β-actin Forward: GTGGGGCGCCCCAGGCACCA 539 55.5Reverse: CTCCTTAATGTCACGCACGATTTC Oct4 Forward: 454 64GGAAAGGCTTCCCCCTCAGGGAAAGG Reverse: AAGAACATGTGTAAGCTGCGGCCC Cdx2-exon 2Forward: GTGTACACGGACCACCAGCG 199 60 Reverse: GGTGGCTGCTGCTGCTGTTGCdx2-exon 1 Forward: AGCCAAGTGAAAACCAGGAC 563 60Reverse: TTTCCTCTCCTTTGCTCTGC Nanog Forward: CTCAGCCTCCAGCAGATGC 200 60Reverse: AGGCATCCCTGGTGGTAGG Eomeso Forward: GGCCACTGCGCGCTACTCC 251 65Reverse: GGCTCCTGGGCCGAACTGC FGF4 Forward: CCTGGTGGCGCTCTCGTTG 199 60Reverse: GCAGGCTGTCGCGGGTGTC fgfr-2 Forward: CACCGTGGCCGTGAAGATG 199 61Reverse: GGGCTCGGAGGTATTCTCG BMP4 Forward: CGCTGGACCCGGGAGAAGC 200 63Reverse: CTCCGGCGTCGGGTCAAGG LIF Forward: CGTGTACCTTGGCACCTCC 199 60Reverse: CCTTACCCGAGGTGTCAGG

CT Std ΔCT qPCR ratio T3ES ACTB 22.85 0.26 hTS ACTB 27.18 0.10 PL ACTB23.82 0.09 hTS-2 ACTB 19.44 0.24 T3ES OCT4 29.22 1.16 −6.37 1.00 hTSOCT4 38.16 0.90 −10.98 0.04 PL OCT4 42.67 0.48 −18.85 0.01 hTS-2 OCT441.08 0.20 −21.64 0.01 T3ES CDX2 35.36 0.59 −12.51 1.00 hTS CDX2 32.530.41 −5.35 143.01 PL CDX2 39.32 0.52 −15.50 0.13 hTS-2 CDX2 40.64 0.86−21.20 0.01 T3ES MIG7 38.35 0.33 −15.50 1.00 hTS MIG7 39.87 0.40 −12.697.01 PL MIG7 35.98 0.16 −12.16 10.13 hTS-2 MIG7 41.22 0.20 −21.78 0.01T3ES IPF-1 30.74 0.39 −7.89 1.00 hTS IPF-1 30.55 0.48 −3.37 22.94 PLIPF-1 30.88 0.19 −7.06 1.78 hTS-2 IPF-1 30.78 0.12 −11.34 0.09

Example 7 Immunocytochemistry

Cultures were fixed with 4% paraformaldehyde for 30 min at roomtemperature and then washed three times with PBS. LSAB kit (Dako,Calif.) was used for immunocytochemical staining as manufacturer'srecommendations. For SSEA-1 and -4 stainings, cells were rinsed withtris-phosphate buffered saline (TBS) and washed with H₂O₂ for 10 min.After blocking the reaction with goat serum (1:200, Dako) for 30 min.Cells then were incubated with primary antibody overnight. After washingthe cells with TBS and treated with streptavidin for 20 min, cells werestained by biotin (20 min), washed again, and treated with 3,3′diaminobenzidine tetrachloride (Boehringer-Mannheim, Mannheim, Germany)for 10 min. Finally, the cells were counterstained with hematoxylinstain. For SSEA-3 staining, similar procedures were followed except thatthe retrieved antigen added, which was obtained using a high-pressurecooker in citrate buffer for 15 min, before washing with H₂O₂. Finally,cells were thoroughly washed with PBS and subjected toimmunofluorescence assays.

Example 8 Immunoprecipitation (IP)

Cells were serum-deprived for overnight and treated with RA (10 μM) for30 min. After pre-clearing with protein G-agarose (Minipore) for 30 min,specific antibodies or IgG were added and incubated overnight. Byincubation with protein G-agarose for 2 h, the beads were washed threetimes with RIPA lysis buffer, boiled in buffer, resolved by 8% SDS-PAGEand immunoblot analysis for various targets as indicated.

Example 9 Flow Cytometry

Cells (5×10⁶ cells/ml) were incubated with a variety of primaryantibodies for 30 min and then incubated with the appropriatefluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- orRho-conjugated secondary antibody (Jackson ImmunoResearch, West Grove,Pa.) at adjusted dilution for 1 h at 4° C. After thorough washing, cellswere re-suspended in PBS (1 ml) and subjected to flow cytometry(FACScan, BD Biosciences, San Jose, Calif.). The data were analyzed withCell-Quest software (BD Biosciences).

Example 10 Microarrays

hTS cells were treated by with or without RA (10 μM) for one- and 5-dayeach. Total RNAs were extracted using TRIzol reagent and subjected forAffymetrix microarray using Affymetrix Human Genome U133 plus 2.0GeneChip according to the manufacturer's proticole (Santa Clara, Calif.,http://www.affymetrix.com) performed at Genomic Medicine Center ofNational Taiwan University College of Medicine, Taipei, Taiwan)

Example 11 Double Immunogold Electron Transmission Microscopy (IEM)

Cells, with or without treatment of RA (10 μM), were examined asdescribed previously (Tsai et al). Briefly, the fixed ultrathin sectionswere pretreated with an aqueous solution of 5% sodium metaperiodate (10min) and washed with distilled water. Grids incubated with an aliquot ofIgG antibody against RXRα (1:50) or Gαq/₁₁ (C-19; sc-392; 1:50) andfollowed by probing with a secondary anti-mouse 6 nm gold particles(1:10; AB Chem, Dorval, Canada) or anti-rabbit IgG 20 nm gold particles(1:10; BB International, UK). Grids were washed with PBS betweenincubation steps and sections blocked by placing the grids on a drop ofPBS with 1% ovalbumin (15 min). After IgG gold, the grids werejet-washed with PBS followed by distilled water. All steps were carriedout at room temperature. Sections were then stained with uranyl acetateand lead citrate and characterized on a Hitachi H-700 model transmissionelectron microscopy (Hitachi Ltd., Japan).

Example 12 Confocal Immunofluorescence Microscopy

Cells were cultured on cover-slips coated with 2% gelatin overnight andtreated with or without RA (10 μM) for 5, 15 and 30 min each. Then,cells were rinsed three times with PBS, fixed with 4% paraformaldehydein PBS for 5 min and permeabilized with 2% FBS containing 0.4% TritonX-100 in PBS for 15 min. This reaction was blocked with 5% FBS at 4° C.overnight followed by incubation with primary antibody RXRα (1:100) orGαq/11(1:100) in PBS at 4° C. overnight. After washing, cells wereincubated with Dye Light 488 or Dye Light 549 conjugated secondaryantibody (1:50; Rockland Immunochemicals Inc., Gilbertsville, Pa.) for 1h. By incubation with DAPI (1:5,000) for 5 min, cover-glass was airdried and sealed for confocal immunofluorescence microscopy (Olympus,Tokyo).

Example 13 Analysis of Unique Population of Human CytotrophoblastsDefined as hTS Cells

Cells obtained from the ectopic chorionic villi were cultured; coloniesformed initially and subsequently proliferated into adherentfibroblast-like cells (FIG. 1a ). Immunocytochemically, these cellsexpressed stage-specific embryonic antigen (SSEA)-1, -3, and -4 (FIG. 1b). These SSEAs-positive cells presented as the same of cytotrophoblastshistologically in the ectopic chorionic villi. However, in the termplacental villi, they appeared mainly at the compartments of villouscore.

To estimate the characteristics of stem cell, the flow cytometricanalyses revealed that these cells expressed high levels of mesenchymalstem cell markers: CD90, CD44, vimentin, and neurofilament, and oftrophoblast marker cytokeratin (CK)-7. They did not expresshematopoietic stem cell markers: CD34 and CD45 and epithelial cellmarkers: E-cadherin, α6-integrin, and L-selectin. They also expressedweakly nestin and CD9 (FIG. 1c ). These facts indicated that thesecytotrophoblasts are distinct from the trophoblastic subpopulationsisolated from mature placental tissues (Aboagye-Mathiesen et al., 1996;Baczyk et al., 2006). Moreover, other supportive evidence included: 1)treatment of these cells with all-trans retinoic acid (RA) resulted in aformation of giant cells (FIG. 1d ) similar to the previous described(Yan et al., 2001); 2) a series of chromosome analyses showed unchangedkaryotypes (see Supplemental FIG. 1a ); 3) subsequent measurement oftelomere lengths confirmed the chromosome stability (see SupplementalFIG. 1b ); and 4) implantation of the cells on the severe combinedimmunodeficient mice created a positive immune chimeric reaction (seeSupplemental FIG. 1c ). Taken all together, these isolated cells likelyrepresent a highly homogeneous population of cytotrophoblasts,exhibiting characteristics of mesenchymal stem cells. Therefore thesecells are regarded as hTS cells.

Example 14 Similarity in Genetic and Biological Characteristics BetweenhTS and hES Cells

To investigate the gene profiling of hTS cells, transcriptase-polymerasechain reaction (RT-PCR) was performed with various primers (seeSupplemental Table 1). The results showed that hTS cells expressed notonly TS cell markers (Cdx2, BMP4, Eomes, and Fgfr-2) but also ES cellmarkers (Oct4, Nanog, Sox2, and FGF4) (FIG. 2a ). The hTS cells weredistinct from PDMS cells (a gift of Dr. C.-P. Chen) in gene distributionby comparing the global gene profiles analyzed by using Affymetrix HumanGenome U133 plus 2.0 GeneChip (Santa Clara, Calif.,http://www.affymetrix.com) (FIG. 2b ).

Interestingly, hTS cells exhibited gene expressions of the three germlayers of ES cells, including: osteopontin, osteocalcin, perlecan,collagen type II, myogenin, myo D1, PPAR γ-2, and adipsin of mesoderm;neurofilament, neurogenin (Ngn)-3, CD133, MAP-2, Neo-D, and nestin ofectoderm; and insulin, Pdx-1, CK-19, somatostatin, Is1-1, Nkx-2.2,Nkx-6.1, and Pax-6 of endoderm (FIG. 2c ). Functionally, hTS cells wereable to differentiate to specialized phenotypes of mesodermal lineage,as seen in hES cells, by using appropriate regimens (In't Anker et al.,2004; Fukuchi et al., 2004; Yen et al., 2005) with modifications (seeSupplemental Table 2), which included osteocytes, chondrocytes,myocytes, and adipocytes (FIG. 2d ). The hTS cells were selectivelyinduced to differentiate into dopaminergic NSCs and insulin-producingislet progenitor cells (see below), as representative of those derivedfrom ectoderm and endoderm, respectively. These results demonstratedthat hTS cells possess both genetic and biological characteristics ofhES cells, which are capable of differentiation into specializedphenotypes of three germ layers.

Example 15 Nanog Maintains Pluripotency of Human Trophoblast Stem Cellsby LIF Withdrawal

The effects of LIF withdrawal on human trophoblast stem (hTS) cells wereexamined since hTS cells expresses pluripotent gene markers of bothembryonic stem (ES) cells and trophoblastic stem (TS) cells such asOct4, Nanog, Sox2, and Cdx2 (FIG. 1a ). hTS cells were treated withdifferent dosages of LIF, i.e., 500 (mimic at ampulla), 250 (mimic atmid-portion), and 125 units (mimic at isthmus) for 3 days each, showingthat LIF promoted Oct4 expression but represses Cdx2, Nanog, and Sox2expressions in a dose-dependent manner (FIG. 1b ). Quantitative PCRanalyses supported these findings (FIG. 1c ). As the relative expressionratio of Oct4 to Cdx2 enables to determine cell fate in early embryonicdifferentiation (Niwa et al., 2000), the Oct4/Cdx2 ratio (0.4-fold)appeared to be the highest at the ampulla which decreased to 0.2-fold atmid-portion and became near one at isthmic portion (FIG. 1d ). Thisdecreasing trend of Oct4/Cdx2 ratio actually facilitates thedifferentiation towards trophectoderm fate (Niwa et al., 2005).Remarkably, a higher Nanog/Cdx2 ratio (2-fold) appeared at cells treatedwith 125 units LIF, while 0.1-fold was noted at 500 units LIF. Theseresults strongly suggested that Nanog as a rescuer of the relativedecreased Oct4 expression is an important determinant for hTS cells tomaintain the pluripotency. This role of rescuer was further supported bythe prominently high Nanog/Oct4 ratio of LIF with 125 units compared tothe ratio of LIF with 500 units and the apparent increase of Cdx2/Oct4ratio at LIF with 125 units (FIG. 1e ) No apparent change of Sox2/Cdx2was found.

Collectively, these results demonstrated that the gradual withdrawal ofLIF concentration from the ampulla toward isthmic portion of humanfallopian tube induces mainly the elevation of Nanog in hTS cells, bywhich it maintains the self-renewal and pluripotent characteristics ofhTS cells mimicking that in mouse ES (mES) cells and human ES cellgrowth without feeder cells. The results indicate that Nanog plays arole in maintaining the pluripotency of hTS cells

Example 16 RA Enhances Nanog Expression

RA is a potent regulator of neuronal differentiation and normally, bybinding to nuclear receptors that interact with retinoic acid responseelements (RAREs) in regulatory regions of target genes (Maden). It hasbeen shown that retinol (vitamin A), a supplier of RA production incell, suppresses cell differentiation mediated by the upregulation ofNanog in ES cells (Chen). Whether or not RA exhibits a similar effect onNanog in hTS cells was examine. The hTS cells were treated with RA forone day and subjected for flow cytometry. The results showed that RApromoted expressions of Nanog, Oct4 and Sox2 but not Cdx2 (FIG. 2f ),which were consistent with the microarray mRNA expression profiling byAffymetrix GeneChip oligonucleotide microarrays (FIG. 2g ). Furthermore,knockout of Nanog with siRNA suppressed RA-induced Nanog, but increasedexpression of Cdx2. In contrast, Cdx2 siRNA promoted Nanog andsuppressed Cdx2 in the RA-induced hTS cells by flow cytometry (FIG. 2h). Taken together, these results indicated that the RA inducesoverexpression of Nanog in hTS cells, by which RA does not change theNanog/Cdx2 ratio in deciding the cell fate.

Example 17 RA Promotes its Receptor RXRα Activation

RA promoted its receptor RXRα activation first in 5 min by Westernblotting assay, however, this action sustained only for 30 min. Instead,an increased RARβ production was observed within 60 min (FIG. 2i ). RAwas observed to interact directly with RXRα and RARβ byimmunoprecipitation assay (FIG. 2j ). Furthermore, the activated RXRαtranslocalized towards the nucleus in a peak at 15 min and henceforth,the nuclear intensity declined by immunofluorescence microscopy (FIG. 2k). The protein Gα_(q/11) subunit was also activated in 30 min (FIG. 2l). To this end, it is likely that RA interacts with RARs at the initialresponsive stage without the assistance of cellularretinoic-acid-binding protein 2 (CRABP-2, FIG. 1d ).

Example 18 RXRα/RARβ Might Belong to the Member of G Protein-CoupleReceptors (GPCRs) Superfamily

This concept was confirmed by observation of direct interaction betweenRXRα and Gα_(q/11) subunit by double immunogold electron microscopy(FIG. 2m ). Next, in order to link the relationship between RXRα/RARβand Nanog, immunoprecipitation assay analysis suggests that RXRα, notRARβ, acts directly on the promoter of Nanog (FIG. 2n ), Further, unlikeES cells, hTS cells contain the major RA generating enzymes:retinaldehyde dehydrogenase type 2 and 3 (RALDH-2 and -3) (FIG. 1d )which enables hTS cells to metabolize retinol into RA. It isdemonstrated that RA acts on hTS cells to produce Nanog by the directinteraction with RXRα/RARβ complex in association with GPCRs to bindwith the promoter of Nanog.

Example 19 RA-Induced Nanog Expression in hTS Cells is Affected by theGradient LIF Content in the Fallopian Tube

The withdrawal of LIF is able to enhance the RA-induced Nanog expressionsignificantly in hTS cells by flow cytometry (FIG. 2i ), suggesting thatthe hTS cell-derived NSCs stand at a position to be able to behave asprogenitor cells by RA induction at the absence of LIF, maintaining themultipotent characteristics for neural subtype specification under anappropriate microenvironmental condition.

Example 20 RA Promotes TH Expression Via a Non-RARE Pathway

These results show that RA induces a nongenomic signaling pathway basedon the initial results that RA stimulated RXR-α, RAR-β and c-Srcexpressions in 5, 120 and 5 min, respectively, in hTS cells measured bywestern blots (FIG. 3a ). To determine whether the RXR-α/RAR-βinteraction belongs to the superfamily of G protein-coupled receptors(GPCRs, double immunogold electron microscopy was used to investigatethe interaction between G-protein Gα_(q/11) and RXR-α. The resultsshowed that RXR-α has a binding interaction with Gα_(q/11) at the cellmembrane (FIG. 3b ) and subsequently, the dissociated Gα_(q/11)stimulates membrane-bound phospholipase C beta (PLCβ) to cleave PIP₂ (aminor membrane phosphoinositol) into two second messengers, IP3 anddiacylglycerol (DAG) (FIG. 3b ).

Subsequently, RA induced a scaffold formation of RXRα, RARβ and [c-Src]by immunoprecipitation assay and using a specific c-Src inhibitor PP1analog (FIG. 3c ).

Example 21 RA Activates the Wnt2B/Fzd6/β-Catenin Pathway

Western blots analyses demonstrated that RA significantly upregulatedWnt2B and proto-oncogene FRAT1 after 4 hr and 24 hr incubation byWestern blots (FIG. 24a ). hTS cells were incubated with RA overnightwith or without siRNA against Wnt2B. Flow cytometric analysis showedthat RA significantly upregulated Wnt2B and its downstream targets,including the mediator protein Disheveled 3 (Dvl3) and proto-oncogeneFRAT1, leading to the inhibitory glycogen synthase kinase-3β (GSK3β),which could be inhibited by knocking down Wnt2B by siRNA (FIGS. 24b and24c ). A similar result was also observed by RT-PCR analysis (FIG. 27).RA also promoted the overexpression of Fzd6 mRNA, member of the Frizzledfamily of 7-span transmembrane receptor (FIG. 24d ). To validate therole of RA in the Wnt2B-mediated expression of Fzd6, we also analyzedthe expression levels of Dvl3 and its downstream effector FRAT1 andshowed that RA-mediated enhancement of Fzd6 could be abrogated by thepresence of siRNA against Wnt2B with a concomitant decrease in GSK3β(FIGS. 24b and 24c ). Subsequently, Western blots analysis showed thatRA significantly activated β-catenin in between 30 min and 24 hr (FIG.24e ). RA induces a novel canonical Wnt2B/Fzd6/β-catenin signalingpathway, allowing the inhibitory GSK3β to stabilize and activatecytoplasmic β-catenin in hTS cells.

Example 22 RA Modulates Histone Deacetylase 6 (HDAC6)

Western blot analysis showed that RA promoted an elevation of Histonedeacetylase 6 (HDAC6), a transcriptional regulation enzyme, in 2 hours,which enabled to directly interact with β-catenin after RA treatment for24 hr by co-immunoprecipitation (IP) assay (FIG. 24f ). Furthermore, weshowed that a nuclear translocation of β-catenin occurred by cellularfractionation assay (FIG. 24g ), supporting the presence of a canonicalWnt2B/Fzd6/β-catenin signaling pathway after RA treatment for 24 hr inthe hTS cells. These observations were further confirmed by the confocalimmunofluorescence microscopy. In the presence of siRNA against HDAC6,nuclear localization of β-catenin was blocked (FIG. 25). Interestingly,we found that a very early expression of β-catenin might appear in 5 minafter RA treatment at the cell membrane (synapse) in the hTScell-derived neuron-like cell. In the nucleus, β-catenin involves intranscriptional regulation by association with transcription factors ofthe TCF/LEF family. Cellular fractionation assay analysis showed thatthis interaction led to the nuclear translocation of β-catenin (FIG. 24e).

Example 23 Interactions Between RARβ and Gβ and Between RXRα andGα_(q/11)

Western blots analysis in hTS cells demonstrated that RA induced rapidproductions of both Gα_(q/11) and Gβ at 30 min and also, retinoid Xreceptor α (RXRα) and retinoic acid receptor β (RARβ) at 30 min and 4hr, respectively, (FIG. 26a ). Analysis of real-time confocalfluorescence microscopy revealed that the GFP-tagged RXRα moved quicklyfrom the cytosolic compartment towards the subcellular regions by RAstimulation within minutes (FIGS. 26b and 26c ), where it co-expressedwith Gα_(q/11) immunocytochemically (FIG. 26d ). This phenomenon wasfurther supported by the double immunogold transmission electronmicroscopy wherein RA stimulated the binding of small gold-tagged RXRαand large gold-tagged Gα_(q/11) at the cell membrane (FIG. 26e ).Biochemically, RXRα physically interacted to Gα_(q/11) and the actionwas inhibited by using RXRα siRNA by IP assay (FIG. 26f ). A similarevent took place between RARβ and Gβ and this action was also inhibitedby using RARβ siRNA by IP assay (FIG. 26g ). IP assay showed a selectivec-Src inhibitor PP1 analog was able to prevent the formation ofRXRα-RARβ heterodimer (FIG. 26h ), suggesting the presence of an unknownmechanism that allowed RXRα and RARβ to function separately. This notionwas further supported by the anchorage of the RA-induced goldparticle-tagged RXRα in the endoplasmic reticulum (ER) observed bydouble immunogold transmission electron microscopy (FIG. 26i ). Takentogether, the data suggest that the RA-induced RXRα and RARβ interactindependently with Gα_(q/11) and Gβ, respectively, at the cell membrane.

Example 24 Akt3/mTOR Signaling and mRNA Translation

Real-time PCR (RT-PCR) analysis and found that RA induced a rapidlytransient elevation of both RXRα mRNA and RARβ mRNA for only 15 min(FIG. 28a ), and a rapid production of RARβ and RXRα within 1 hr (FIG.26a ). Focus on examining whether subcellular mRNA localization of RXRαwas involved in these cellular processes based on the facts that thereis enrichment of mRNA in axonal growth core and its association withmRNA localization in neurons and the RA-enhanced RARα levels mediatelocal GluR1 synthesis in the dendritic RNA granules, contributing to theRARα-modified translation for synaptic formation at neuronal membrane.Subsequently, IP assay showed that RA induced binding between Gβ andphosphatidylinositol 3-kinase (PI3K) (FIG. 26g ) and activated PI3K withits downstream effectors all Akt isoforms, including Akt1 and Akt2 inbetween 30 min and 4 hr as well as a transient Akt3 in 1 hr by Westernblots analysis (FIG. 28b ). After treatment with RA for 24 hr, allexpressions of Akt isoforms were inhibited by pretreating PI3K inhibitorWortmannin by flow cytometry (FIG. 28c ) and RT-PCR analysis (FIG. 29a), indicating the presence of Gβ/PI3K/Akt signaling. Notably, Akt hasbeen recently emerged as a crucial regulator of neurite outgrowth topromote neuronal survival, the RA-induced Akt3 (4 hr) could bind to themechanistic target of rapamycin (mTOR), which was inhibited by siRNAagainst Akt3 (FIG. 28d ), leading to a temporal phosphorylation of mTORat site serine 2448 in 4 hr detected by using specific antibody (CellSignaling Technology). However, this action disappeared after 24 hrincubation (FIG. 28e ). This function was inhibited by knockdown of Akt3using siRNA by Western blots (FIG. 28f ) and by flow cytometry (FIG. 29c). Immediately, Western blots analysis showed that by RA treatment for 4hr, phosphorylated mTOR interacted directly with eukaryotic translationinitiation factor-4E binding protein 1 (eIF4EBP1) (FIG. 28g ) andactivated eIF4EBP1 (FIG. 28h ). Knockdown of phosphorylated mTOR byusing siRNA, phosphorylation of eIF4EBP1 was inhibited; insteadphosphorylation of elongation initiation factor 4E (eIF4E) was activated(FIG. 28h ), implicating that a dissociation of eIF4E from theeIF4E/eIF4EBP1 complex occurred. Phosphorylation of eIF4E enables tocause cap-dependent translation of mRNA. Overall, these observationsexplain how RA enables to induce subcellular mRNA translation throughthe activation of RXRα mRNA and RARβ mRNA to locally produce RXRα andRARβ, respectively, because knockdown of eIF4E by siRNA bothinteractions between RXRα and Gα_(q/11) and between RARβ and Gβ wereinhibited by IP assays (FIG. 28i ). These results support that Akt3/mTORsignaling plays as an initiator of local synthesis of RXRα and RARβ.Although RA stimulated elevation of elongation initiation factor 4B(eIF4B), this action was not influenced by siRNAs against either mTOR or4EBP1, suggesting another mechanism in regulating eIF4B expression (FIG.28h ). The spatiotemporal Akt3 promotes subcellular localization forRXRα and RARβ productions via mTOR signaling.

Example 25 CREB1 on the Mainstream in Dopaminergic Specification

Gβ/PI3K downstream effector Akt1 directly binds and activates cAMPresponsive element binding protein 1 (CREB1) through phosphorylation atserine 133 site (FIG. 30a ). The interaction of Akt1 and CREB1 wasinhibited by Akt1 siRNA (FIG. 30b ). The phosphorylated CREB1 targetedand transcribed dopamine precursor tyrosine hydroxylase (TH) gene bychromatin immunoprecipitation (ChIP) assay (FIG. 30c ), which wasinhibited by CREB1 siRNA (FIG. 30d ). To this end, results suggestedthat the RA-induced RARβ/Gβ/PI3K/Akt1/CREB1 pathway played a role in theTH transcription in dopaminergic neurogenesis. To support this notion invivo, a model with 6-OHDA-induced PD rats who received intracranialtransplantation of the hTS cell-derived trophoblastic NSCs (tNSCs) atthe lesioned striatum was used. Examination of the brain sections at12-week postimplantation revealed that in the substantia nigra compacta,co-expression of CREB1 and TH was observed in the newly dopaminergic(DA) neurons in the newly dopaminergic (DA) neurons in the therapeuticside compatible with that in the normal side by immunofluorescencetissue analysis (FIG. 30e ). Both TH and CREB1 activities were higher inthe regenerated DA neurons compared to that normal ones (FIG. 30f ).Interestingly, an apparent CREB1 expression was observed in the nucleousof DA neurons. These findings may explain why CREB1-deficient mice aresusceptible to neurodegeneration.

Example 26 Study of RXRα/Gα_(q/11) in ER Calcium Regulation

Western blots analyses in between 30 min and 4 hr showed that RA inducedgradual activation of Gα_(q/11) that triggered the catalysis of themembrane-bound phospholipase C (PLC-β), leading to the degradation ofmembrane phosphoinositol PIP2 (FIG. 21a ) to produce second messengerinositol (1, 4, 5) triphosphate (IP3) consistent with the conventionalGα signaling described previously. IP3 activated its receptor IP3R (FIG.21a ) located at ER, causing intracellular calcium elevation (FIG. 21b). To ascertain the origin of intracellular calcium, cells were culturedin the calcium-free medium wherein RA induced a transientlyintracellular Ca²⁺ release by real-time live cell immunofluorescencemicroscopy (FIG. 21b-a ). The depletion of ER calcium level could berescued by adding extrinsic CaCl₂ for homeostasis and cell protection,exhibiting a pattern of the store-operated calcium entry (SOCE). Theprocess of calcium release in the ER was inhibited by IP3R specificinhibitor 2-APB, in a dose-dependent manner (FIG. 21b-b ). These resultsindicate that the ER-released intracellular calcium elevation isresponsible for the RA-induced Gα_(q/11) signaling pathway in hTS cells.

KCl could activate L-type calcium channels after the RA-induceddepletion of ER calcium in the calcium-free medium in hTS cells (FIG.21b-c ). The L-type calcium channel antagonist nifedipine was able toblock this signaling (FIG. 21b-d ). RA regulation of intracellular ERcalcium was associated with L-type calcium channels.

Example 27 Investigation of CaMKII in Excitation-Neurogenesis Coupling

Western blot analysis indicated that RA induced a spatiotemporalactivation of CaMKII in 1-2 hr (FIG. 21a ). Immunoprecipitation assayanalysis demonstrates that CaMKII directly phosphorylated and activatedCREB1 (FIG. 21c ) compatible with the previous study that CaMKII encodesL-type calcium channel activity locally to signal to nuclear CREB inexcitation-transcription coupling. Western blot analysis indicated thateukaryotic initiation factor 4B eIF4B siRNA inhibited expressions ofCaMKII, calcineurin, and eIF4B (FIG. 21d ). Axons contain a variety ofmRNA encoding specific protein synthesis locally, including CaMKII,calcineurin, and CREB1 in developing neurons. CREB1 enables theretrograde trafficking for specific transcriptional processes in thenucleus responsible for the signal of distal axons. The extrinsicRA-triggered local protein synthesis of CaMKII can be inhibited by eIF4BsiRNA in hTS cells. Therefore, this locally activated CaMKII signalbehaved similarly to CREB1, suggesting a rapid inducible genetranscription upon extracellular cues.

The transient CaMKII bound and activated eukaryotic initiation factor 4B(eIF4B) (FIG. 21c ) to initiate mRNA translation machinery via acap-independent mechanism. Western blot analysis indicated that thisaction was inhibited by a selective CaMKII inhibitor KN93 after RAtreatment (FIG. 21e ). This CaMKII/eIF4B signaling then integratedeIF4B/c-Src/Nanog signaling pathway to accomplish the signaling pathwayfrom RXRα/Gα_(q/11) to Nanog for self-renewal and proliferation oftNSCs. These results first explored that the Gα_(q/11) signal-derivedCaMKII excitation was involved in the maintenance of self-renewal oftNSCs.

Western blot assay and immunoprecipitation assay analyses demonstratedthat CaMKII binds to and activates parkinson protein 2 (parkin) (FIGS.21a and 21f ). In turn, parkin directly interacted and activatedmicrotubule-associated protein tau (MAPT) (FIGS. 21a and 21f ), which ispreferentially located in axons and stimulates microtubule assembly.Consequently, MAPT directly bound to SNCA (FIGS. 21a and 21g ) to form aparkin/MAPT/SNCA complex. Where MAPT interact and activate tubulin(FIGS. 21a and 21h ), a microtubule element expressed exclusively inneuron that stabilizes and promotes microtubule assembly. Together,these results suggested the importance of axonal behaviors in earlyneurogenesis.

Example 28 Activation of Calcineurin/NFAT1 Signaling

Western blot assay analysis demonstrated that RA induced production ofcalcineurin (FIG. 21a ). Pretreatment with 2-APB inhibited Calcineurin,NFAT1, and MEF2A expression (FIG. 21i ), linking the ER calcium andcalcineurin molecules. Calcineurin immediately dephosphorylated NFAT1, akey regulator of T cell activation and anergy, showing a transientfashion in 30 min to 2 hr (FIG. 21a ). This action was also inhibited by2-APB as evidenced by immunoprecipitation assay analysis (FIG. 21h ),linking the ER calcium to calcineurin/NFAT1 signaling. Moreover, RAinduced a transient interaction of NFAT1 and importin, anucleocytoplasmic transporter (FIGS. 21a and 21j ), leading to the NFAT1nuclear translocation by cell fractionation assay (FIG. 21k ). Thistemporal effect of NFAT1 is thought to be one mechanism by which cellsdistinguish between sustained and transient calcium signals.

Example 29 Study of Wnt and G Protein Signaling Pathways

The inhibitory GSK3β (at serine/theronine site) of canonical Wntsignaling maintained stabilization of cytoplasmic β-catenin aftertreating RA overnight but with a slightly decreased levels in 30-120 min(FIG. 24d ). Unexpectedly, among Akt isoforms Akt2 was able to bindGSK3β in 4 hr (FIG. 21l ); however, flow cytometric analysis showed thatGSK3β was initially activated in 4 hr but transited into inhibitorylater by RA treatment overnight (FIG. 21m ). This phenomenon was furtherconfirmed by using Akt2 siRNA (FIG. 21n ). To explain this functionaldivergence, it was confirmed that the initial activation of GSK3β wasdue to the phosphorylation at Tyr216 site by Akt2 followed theinhibition was due to the phosphorylation at serine/theronine site (FIG.21m ). These results demonstrate that site-specific phosphorylation ofGSK3β by various protein kinases determines the fate of downstreameffector. Moreover, active GSK3β phosphorylated MAPT via directinteraction (FIG. 21h ). In turn, MAPT interacted with and activatedtubulin (FIGS. 21a and 21h ) to promote microtubule assembly. Notably,the conversational bridges among Wnt2B, Gβ, and Gα_(q/11) signalingpathways are constructed during early neurogenesis.

Example 30 Study of Transcription Factors for Dopaminergic Neurogenesis

In the nucleus, interaction of β-catenin and CREB1 represented amainstream in TH transcription (FIG. 30a ). Active β-catenin, in turn,bound to lymphoid enhancer factor 1/T cell factor 1 (LEF1) (FIG. 22a ),leading to the switch of LEF1 from repressor to activator oftranscription. LEF1 then recruited and interacted with Pitx2, member ofa superfamily of bicoid-related factor (FIG. 22a ). Whereas LEF1promoted Pitx2 gene transcription but not Pitx3 gene by chromatinimmunoprecipitation (ChIP) assay (FIG. 22b ) compatible with thatβ-catenin, Pitx2, and LEF1 interact to synergistically regulate theLEF-1 promoter.

Furthermore, the transient nuclear active NFAT1 plays as transcriptionfactor to produce cytokines and TNF-α for immune responses. However,this action was unlikely to occur in the present case because thephosphorylated GSK3β enables to inhibit the DNA binding ofcalcineurin-induced NFAT1 in the nucleus and to promote nuclear export.Therefore, active cytoplasmic NFAT1 would interact and activatecytoplasmic transcription factor myocyte enhancer factor 2A (MEF2A)(FIGS. 22c and 22d ) because this action was inhibited by NFAT1 siRNA(FIG. 22e ). Notably, the rapid inducible CREB1 entered the nucleus andtranscribed MEF2A gene that produced MEF2A protein (FIG. 22f ). MEF2Amight function in multiple ways at gene transcription (FIG. 22g ),including transcription itself via auto-regulation to produce moreMEF2A, transcription TH gene for dopaminergic specification,transcription SNCA gene for SNCA/MAPT/parkin complex formation, andinteraction with EP300 and Pitx2, which was inhibited by MEF2A siRNA(FIG. 22h ).

The active ER300 not only targeted HDAC6 gene but also TH gene by ChIPassays (FIG. 22i ). HDAC6 then enabled to carry β-catenin for nucleartranslocalization (FIGS. 24e and 24f ). Taken together, an executivetranscription complex was formed and destined for TH gene transcription.Among them, CREB1, EP300, and MEF2A were able to directly targetpromoter of TH gene while β-catenin, LEF1, and Pitx2 performed asco-activator of enhancer during transcription processes. Western blotsanalysis show the various molecular activities at 4 hr and 24 hr (FIG.22j ).

Example 31 Animal Studies

For animal study, reporter cells were prepared by transfecting theF1B(−540)-GFP and pSV2neo plasmids into hTS cells followed by selectionwith G418. Greater than 95% of hTS cells showed co-expressions ofF1B-GFP and TH-2. Second, Parkinson's disease was induced in “young”Spraque-Dawley rats (n=12, body weight, 225-250 gm) by injectingneurotoxin 6-hydroxydopamine (6-OHDA) into rat brain unilaterally asdescribed below.

All experiments were conducted and performed according to the guidelinesof the ethical board of the Institutional Review Boards of the Hospital,Kaohsiung Medical University Hospital and Ethical Committee at MedicalCollege of National Chung Kong University, Tainan, Taiwan.

Induction of Parkinsonism

Twelve Sprague-Dawley rats (560+65 g (pre), 548+46 g (post) of bodyweight) were used as model for 6-OHDA-lesioned hemiparkinsonism (Javoyet al., Brain Research, 102:201-15, 1976). For surgery, after anesthesiaby chloral hydrate (4%, 1 cc/100 g of body weight), stereotaxic lesionswere carried out by infusion of 6-hydroxydopamine (Sigma) into the rightmedian forebrain bundle (AP 2.8/Lat 2.2/Dep 8.0 mm) at a rate of 1μg/0.5 μl/min for 8 min (injection pump: CMA 100). After 10 min, thetube was removed. Two weeks later, apomorphine-induced rotation wastested in a plastic bowl (36 cm in diameter) 20 min after receivingapomorphine injection (25 mg/kg) subcutaneously. The contralateralturning rotation was monitored and recorded for 20 min using a videocamera. Rats with the number of rotations over 25 per 5 min wereeligible for the study. For cell transplantation, cells weretransplanted into two sites (each site: 3×10⁶/4 μl) within the rightunilateral striatum (1st site: AP+1/Lat+2.7/Dep 6.4 mm; 2^(nd) site:AP+0/Lat+2.7/Dep 6.4 mm). The control group was given PBS with the sameapproach. Apomorphine-induced rotation was measured at 0, 3, 6, 9, and12 weeks after cells injection. The results were expressed ascontralateral turns/5 min (FIG. 5A).

In order to examine the effects of NSCs induced by different time of RA,the eligible rats were randomly divided into three groups: the one- andthe 5-day RA-induction groups and the control. Before transplantation,hTS cells were transfected with F1B-(−540)-green fluorescent protein(GFP) and pSV2neo recombinant plasmid DNA followed by G418 selection toachieve a yield over 95%. Each rat received GFP-tagged NSCs with 6×10⁶cells in total and the control one received phosphate-buffered saline asvehicle. The therapeutic effect was assessed by apomorphine-inducedrotation test (Iancu et al., 2005) every 3 weeks after implantation.

Experiment 1

Adult Sprague Dawley rats (BW: 225-250 g) were used as graft recipientsand housed on a 12 h light/dark cycle with ad libitum access to food andwater. The lesioned rats (n=12) were first divided into three groups:(a) lesioned and transplanted with one-day RA-induced NSCs (n=4), (b)lesioned and transplanted 5-day RA-induced NSCs (n=4) and (c) lesionedand non-transplanted control (n=4). Rats were anesthetized by Zoletil(50 mg/kg, s. c., Virbac Lab. Carros, France) and the lesioned rats wereunilaterally injected with 6-OHDA (8 μg/4 μl in 0.1% 1-ascorbicacid-saline; Sigma-Aldrich, Mo.) into the left MFB (AP 2.8, Lat 2.0, Dep8.0 mm) and SN (AP 5.0, Lat 2.2, Dep 7.5 mm) according to bregma anddura in mm and awaiting for 10 min at the site. Transplantation of thehTS cell-derived NSCs (1×10⁶ cells/5 μl/5 min) into the DA-depletedstriatum at two sites (AP+1.0, Lat+2.7, Dep 6.4 and AP+0, Lat+2.7, Dep6.4) and the cannula was left in place for 5 min before slowlyretracting it. The cell viability remained stable between 96 and 98%during the implantation procedure. Sham rats received vehicle withoutcells. Lesion was evaluated by means of apomorphine-induced rotationevery one week after the 6-OHDA lesion to achieve a stablehemiparkinsonian status (>300 rotations/h). Graft effect was assessedevery 3 weeks by apomorphine-induced rotation test until 12 weeks. At 18weeks postimplanation, rats were sacrificed and brain sections weresubjected for TH-DAB immunostaining.

Experiment 2

The PD rats were controlled at pre-test with 560+/−65 g and post-test at548+/−46 g in body weight. The lesioned rats (n=16) were created as inthe experiment 1 and divided into two groups: (a) lesioned andtransplanted with cells (n=8) and (b) lesioned and transplanted withoutcells as control (n=8) by transplantation with one-day RA-induced NSCs.Cells were grafted by injection at AP+1.0, Lat+2.7, Dep 6.4. Behavioralassessments were carried out every 3 weeks until 12 weekspostimplantation as described below. At 13 weeks, all rats weresacrificed and the brain sections were subjected for TH-DABimmunostaining and the TH-positive cells were analyzed by densitometry.

Behavioral Assessments

Locomotor Activity Assays.

For rats, spontaneous locomotor activity was monitored in a circularcorridor (10 cm wide and 60 cm in diameter with walls 30 cm high; MedAssociates Inc., St Albans, Vt.). Four photoelectric cells locatedequidistantly around the walls of the circles detected an animal'shorizontal ambulatory activity by way of beam interruptions. Data wererecorded via a PC equipped with customized software (Med Associates).Separate groups of animals were tested with 10 mg/kg (n=6 per group) and20 mg/kg (n=12 per group) cocaine. Animals were randomized intotreatment groups (HSV-LacZ and HSV-RGS9-2) and habituated to thelocomotor apparatus for 2 hr. On the next day, animals received HSVvectors in the nucleus accumbens shell on a stereotaxic frame. Following2 days of recovery, animals were tested with cocaine on locomotoractivity for 2 hr. Data were analyzed by two-way ANOVA (HSV×time) withBonferroni post hoc test.

For mice, locomotor activity was determined in an automated system inwhich the activity chambers were plastic cages (12×18×33 cm) with 10pairs of photocell beams dividing the chamber into 11 rectangular fields(Hiroi et al., 1997). Mice were tested at the same time each day by anexperimenter who did not know the genotype of the mice. For acuteexperiments, animals were habituated to the chambers for 30 min, afterwhich time they received i.p. injections of saline or varying doses ofamphetamine, cocaine, or apomorphine, and locomotor activity wasassessed for an additional 30 min. For chronic experiments, animals wereplaced in the chambers immediately after an i.p. saline injection on thefirst 3 days. Horizontal activity was then measured for 10 min. On days4-8 (C1-C5), animals were given cocaine (7.5 mg/kg i.p.) and activitywas measured for 10 min. The short time periods used for rats and micehave been shown in previous studies to avoid the potentially confoundingeffects of stereotypy in measures of ambulatory locomotor activity.

Three behavioral tests were performed: (i) drug-induced rotation toassess lesion and graft effects, (ii) footprint analysis to evaluatehind limb gait patterns, and (iii) the ladder rung walking test toassess skilled walking performance (hind limb/forelimb coordination andpaw placing accuracy).

Apomorphine-Induced Rotation Test.

Briefly, rat was placed in a large round chamber (16 cm in diameter) fora period of 40 min after apomorphine administration subcutaneously (0.5mg apomorphine in 0.01% ascorbic acid in 0.9% normal saline/kg bodyweight, Sigma-Aldrich). All rotations were recorded on the videotape andthe net rotation asymmetry was calculated. Data were calculated asnumbers of total turn in 30 min. Data were analyzed by using Matlabsoftware.

Apomorphine-induced rotation (apo) was also observed for 60 min afterintraperitoneal injection of 0.5 mg/kg apomorphine solution(Sigma-Aldrich, 0.5 mg apomorphine in 0.01% ascorbic acid of 0.9% normalsaline). Rotational bias was assessed in rotometer boxes after thelesion (2 and 3 weeks post LX) and after the transplantation (3 and 6weeks post TX) as described previously ([59]; FIG. 2). Data of the 2weeks post LX and 3 weeks post TX drug-induced rotations are not shown.Three days later amphetamine-induced rotation (amph) was carried out for90 min after intraperitoneal injection of 1 ml/kg amphetamine solution(Sigma-Aldrich, Steinheim, Germany: 2.5 mg d-amphetamine per 1.0 mlsaline). Five animals were excluded from the study because they showed<4.0 full body turns contralaterally to the lesioned side afterapomorphine injection and <6.0 full body turns ipsilaterally to thelesioned side after amphetamine injection. Apomorphine-induced rotationis presented as net rotation in negative values, and amphetamine-inducedrotation is presented as net rotation in positive values.

Drug-induced rotation after the injection of apomorphine (A) and theinjection of amphetamine (B). The rotational bias is shown as the totalamount of full body rotation. The dollar sign ($) indicates asignificant difference between the sham and the tx rats. pre TX=6 weeksafter the lesion, post TX=6 weeks after the transplantation. Note thatthere were significant graft effects (reduction of rotational bias afterapomorphine injection; overcompensation after amphetamine injection).

Bar Test for Akinesia.

For the bar test, rat was placed gently on a table with a posture thatboth the contralateral and ipsilateral forepaws were placedalternatively on a horizontal acrylic bar with 0.7×9 cm in size. Thetime from placing of forepaws to the first complete removal of each ofthem from the bar was recorded. Total time spent by each paw on theblocks was recorded as described previously (Fantin).

Footprint Analyses (Spatiotemporal Gait Analyses).

Footprint analyses including walking speed, step length, stride lengthand base of support were performed to evaluate hind limb walkingpatterns as described previously (Klein). The rats had to walk on aplastic board through a walkway (50 cm long, 8 cm wide). The parametersincluding stride length, limb rotation (angle between a virtual linethrough the third digit and the centre of the palm and a virtual lineparallel to the walking direction) and distance between feet (distancebetween feet of the left and right stepping cycle) with five sequentialsteps were recorded by a video camera (Casio EX-F1, Japan) and analyzedby Matlab software.

Ankle joint rigidity assessment is evaluated using suitable methods.Suitable Electrophysiological assays are used to determine %dopaminergic neuron recovery in the brain.

Immunohistochemistry

For TH immunohistochemistry, the animals received a terminal dose of 60mg/kg sodium pentobarbitone i.p. (Apoteksbolaget, Sweden) and weretrans-cardially perfused with 50 ml saline (0.9% w/v), followed by 200ml ice-cold paraformaldehyde (4% w/v in 0.1 M phosphate bufferedsaline). The brains were removed, post-fixed for 2 h in 4%paraformaldehyde and cryo-protected overnight in sucrose (25% w/v in 0.1M phosphate buffered saline) before being sectioned on a freezingmicrotome (Leica). Coronal sections were collected in 6 series at athickness of 20 μm.

Immunohistochemical procedures were performed as follows. Free-floatingsections were incubated with primary antibodies overnight at roomtemperature in an incubation solution of 0.1 M phosphate buffered salinewith potassium containing 5% normal serum and 0.25% Triton X-100(Amresco, USA). Secondary antibodies were diluted in phosphate bufferedsaline with potassium containing 2% normal serum and 0.25% Triton X-100and applied to the original solution for 2 h at room temperature.Detection of the primary-secondary antibody complexes was achieved byperoxidase driven precipitation of di-amino-benzidine, or conjugation ofa fluorophore (either directly to the secondary antibody or with astreptavidin-biotin amplification step where necessary). For detectionof c-Fos, nickel sulphate (2.5 mg/ml) was used to intensify thestaining. Slide mounted sections labeled with fluorescent markers werecover-slipped with polyvinyl alcohol-1,4-diazabicyclo[2.2.2]octane anddi-amino-benzidine labeled sections were dehydrated in alcohol andxylene and cover-slipped with DePeX mounting media (BDH Chemicals, UK).Primary antibodies and dilution factors were as follows: mouseanti-Calbindin_(28KD) (1:1000: Sigma), rabbit anti-c-Fos (1:5000,Calbiochem), chicken anti-GFP (1:1000; Abcam), rabbit anti-GFP (1:20000; Abcam), rabbit anti-GIRK2 (1:100; Alomone Labs, Jerusalem, Israel)rabbit anti-PITX3 (1:100; Invitrogen) and mouse anti-tyrosinehydroxylase (TH: 1:4000; Chemicon). Secondary antibodies, used at adilution of 1:200, were as follows: (i) direct detection-cyanine 3 orcyanine 5 conjugated donkey anti-mouse, cyanine 2 conjugated donkeyanti-chicken, cyanine 5 conjugated donkey anti-mouse (JacksonImmunoResearch); and (ii) indirect with streptavidin-biotinamplification-biotin conjugated goat anti-rabbit or horse anti-mouse(Vector Laboratories) followed by peroxidase conjugated streptavidin(Vectastain ABC kit, Vector laboratories), or cyanine 2/cyanine 5conjugated streptavidin (Jackson ImmunoResearch).

In Vivo Study on CREB1 Expression in Dopaminergic Specification

To obtain the brain sections, rats were anesthetized by sodiumpentobarbitone (60 mg/kg i.p., Apoteksbolaget, Sweden) andtrans-cardially perfused with saline (50 ml, 0.9% w/v) followed byice-cold paraformaldehyde (200 ml, 10% w/v in 0.02 M PBS) were performedat 18- and 12-week in the acute and chronic PD rats, respectively. Brainsections were subjected for immunocytochemistry, immunohistochemistry,and immunofluorescence tissue analysis as indicated.

The 6-OHDA-induced PD rats who received intracranial transplantation ofthe hTS cell-derived trophoblastic NSCs (tNSCs) at the lesioned striatumwere examined to investigate CREB1 expression. Examination of the brainsections at 12-week postimplantation revealed that in the substantianigra compacta, co-expression of CREB1 and tyrosine hydroxylase (TH) wasobserved in the newly dopaminergic (DA) neurons in the therapeutic side,compatible with that in the normal side by immunofluorescence tissueanalysis (FIG. 30e , insert). Both TH and CREB1 activities were higherin the regenerated DA neurons compared to that normal ones (FIG. 30f ).An apparent CREB1 expression was observed in the nucleus of DA neurons.These findings can assist in the explanation of how CREB1-deficient miceare susceptible to neurodegeneration

In Vivo Study on Regeneration of the Dopaminergic Nigrostriatal Pathway

To further verify the regeneration of the dopaminergic nigrostriatalpathway after cell therapy, immunofluorescence tissue analysis wasperformed (TissueGnostics Gmbh, Vienna, Austria). Brain sections wereinvestigated, including 14 acute PD rats (i.e., 2 at 1-week and 2 at6-week post-injury and 2 controls, 6 at 12-weeks after celltransplantation and 2 controls) and 4 chronic PD rats (i.e., 2 at12-week after cell therapy and 2 controls). In the SNC, 6-OHDA causedprogressive neural degeneration, resulting in various sizes of cavity at6 weeks post-injury (FIG. 31). Intriguingly, after tNSCs therapy,numerous DA neurons appeared at the wall of the cavity with TH-positivenervous terminals projecting into the cavity (FIG. 31, insert).Quantitative analysis showed that the number of DA neurons reducedapparently to 48% and 13% at 1- and 6-week post-injury in the SNC,respectively, compared to the intact side (FIGS. 32a and 33).Remarkably, the loss of DA neurons could be reduced by up to 67% aftertNSCs therapy.

While in the striatum, DA neurons reduced to 78% and 4% at 1- and 6-weekpost-injury, respectively (FIG. 32a ). Similarly, the lost DA neuronscould be regenerated by up to 73% after tNSCs therapy. Consistent withobservations (FIG. 6), DA neuronal circuitries were well-established inthe therapeutic side of SNC similar to the intact sideimmunohistochemically (FIG. 32b ). The recovery rate of DA neuronscounted for 78.4±8.3% (mean±SEM; n=4) in the SNC (FIG. 32c ) compatiblewith the 67% in the immunofluorescence analysis (FIG. 23a ).

Since glial cells play as mediators in guiding the migration of neuronsto their destinations or as sources of neural regeneration, 6-OHDAcaused not only degeneration of both DA neurons and GFAP(+) cells butalso disarrangement of the striato-pallido-nigral axons in the striatum(pencils of Wilson). These phenomena were clearly improved after tNSCtherapy, showing numerous GFAP(+) cells embedded in the fine myelinatedfibers (FIG. 32d ). As noted, the GFAP(+) cells regenerated from 65.5%at 6 weeks post-injury to 93.9% after tNSC therapy in the lesionedstriatum (FIG. 32e ). This fact might reflect astrocytic activation,attributable to the implanted tNSC subtypes, i.e., GRP and astrocytes.These results indicate that transplantation of tNSCs regenerates thedopaminergic nigrostriatal pathway in chronic PD rats thereby explainingthe improvement of behavioral deficits. Optimizing the regeneration ofDA neurons would continue for at least 18 weeks postimplantation basedon the retention of tNSCs in the lesioned pathway (FIG. 5).

In vivo, hTS cells were implanted into male severe combinedimmunodeficient (SCID) mice intramuscularly for 6-8 weeks.Histologically, no teratoma was found; but minor chimeric reaction withmyxoid-like bizarre cells was observed between the muscle fibers (FIG.7H). These results reveal the advantage of hTS cells and tNSCs intranslational medicine compared to hES cells with respect to teratomaformation.

Statistics

All data are expressed as mean±SEM. Differences were assessed by usingrepeated measure analysis of variance (ANOVA) tests (SPSS Release 12.0software) and applied least significant difference test (LSD) post hoccomparisons after repeated measure ANOVA tests between two groups forapomorphine-induced rotation analyses. Student t test, paired t test wasused when appropriate. p-value <0.05 was considered significant.

The animal experiments show that tNSCs injected into the lesionedstriatum are able to migrate upstream to subnigral nucleus vianigrostriatal pathway evidenced by GFP-tagged immunofluorescence studyafter 18 weeks implantation. Second, the efficacy in improvingbehavioral deficits is higher than expected, for example, recovery ofdopaminergic neurons 12 weeks post-implantation is 28.2%. Third, thereis neither immunosuppression nor tumorigenesis observed. Further, theimprovement in 28.2% dopaminergic neurons and behavioral deficits ismaintained in a chronic PD rat over one year after 6-OHDA induction.These results indicated that transplantation of tNSCs was able toregenerate the dopaminergic nigrostriatal pathway and functionallyimprove the behavioral impairments in acute PD rats.

Chronic PD Animal Model

To more closely mimic the pathologically progressive nature of PDpatients, a chronic PD rat model was developed by breeding methods overone year (12.3 months in average). The apomorphine-induced rotation testwas performed monthly to ascertain the rats' PD state throughout theexperiment. Group I (n=6) received tNSCs while group II was the control(n=6). Behavioral assessments were performed every 3 weeks, includingthe apomorphine-induced rotation test, the bar test for akinesia, thestepping test for rigidity, and the footprint analyses for posturalimbalance and gait disorder.

In Group I, a significant improvement of the apomorphine-inducedcontralateral rotations was achieved from 3 weeks to 12 weekspostimplantation similar to the previous study in acute PD rats (FIG.6A). The bar test showed that the grasping time of the affected forelimbwas significantly shortened at 3 weeks, and continued to improve at 12weeks (FIG. 6B). All assessments by step length (FIG. 6C), stride length(FIG. 6D), walking speed (FIG. 6E), and base of support (FIG. 6F)revealed significant improvement from 3 weeks to 12 weekspostimplantation. These studies were performed on a well-designedwalkway (FIG. 6G). These results indicated that transplantation of tNSCswas able to regenerate the dopaminergic nigrostriatal pathway andfunctionally improve the behavioral impairments in chronic PD rats.

Example 32 Pull and Push Mechanism

G protein-coupled receptors (GPCRs) communicate between internal andexternal environments and couple with heterotrimeric G proteins at thecell membrane. However, the mechanisms that explain how the activatedGPCRs initiate this process are less clear. A recent report has shownthat upon the introduction of ligand, both Gα₁₃ and Gα_(q/11) subunitsinteract with AhR-interacting protein where Gα₁₃ leads to thedestabilization, translocation and ubiquitination of cytosolic AhR. Therole of G protein signaling in the nongenomic AhR pathway was explored.BBP was chosen as an exogenous ligand and COX-2 as an activated target,as COX-2 causes inflammation, metabolism and carcinogenesis in a varietyof human cells, including hepatic cancer cells.

Immunofluorescence studies are considered important for the dynamicstudy of signal transduction through their ability to capture snapshotsof molecular changes in the cell. Human hepatic Huh-7 cancer cells werepre-transfected with pGFP-C1-AhR by using LT1 transfection reagent(Mirus Bio LLC, WI) and total internal reflection fluorescencemicroscopy to selectively observe the molecular events in thecytoplasmic region immediately beneath the plasma membrane. When BBP wasintroduced, a rapid but transient recruitment and translocation of theGFP-tagged AhR occurred at the subcellular membrane regions, showing afast elevation and peaking in 115 seconds followed by a gradual decreasein AhR that occurred over a few minutes (FIG. 14a ). This fast dynamicmovement of the memAhR at the subcellular membrane is reminiscent of thenotion of soft-wired signal transduction. AhR has been found to serve anadaptive function through its regulation of biotransformation enzymesand change in localization within the cell, triggering its ownactivation.

Next, the association between BBP and AhR was examined by reversetranscription polymerase chain reaction (RT-PCR). BBP significantlyinduced mAhR expression in 5 min, peaking at 15 min and graduallyreturning to a slightly higher constitutive steady-state (FIG. 14b ).Interestingly, Western blot analysis showed the BBP-induced elevation inAhR production at 15 min, slightly decreased production at 30 min, and are-elevation at 1 h (FIG. 14c ). The different patterns of AhRexpression at these time-points found in these two assays can beexplained by the differences between subcellular mRNAs activation andconstitutive synthesis, supporting the notion of “cytoskeleton in mRNAtransport”. Therefore, it is likely that Huh-7 cells contain thestructural machinery of mRNA needed for local protein translation inresponse to exogenous stimulation²¹ and is called memAhR hereafter. Thelower mRNA level probably represents the constitutive AhR activity inthe maintenance of differential stability of cells. Upon ligandactivation, heterotrimeric G proteins can dissociate into Gβγ dimers andGα subunits, including G_(s), G_(i), G_(q/11) and G_(12/13), eachperforming different functions. BBP induced both of Gα_(q/11) and Gβproduction in 30 min (FIG. 14d ). The elevation of Gα_(q/11) was due tothe direct interaction between memAhR and Gα_(q/11) (FIG. 14e ). Theseresults were further confirmed by knockout of AhR using siRNA in cells(FIG. 14f ). Clearly, these data indicate that by BBP stimulation, theGPCR was excited and led to the dissociation of heterotrimeric Gαβγ intoGα and Gβγ subunits, enabling Gα_(q/11) to interact with their upstreamactivator, memAhR. Because AhR has been associated with Gα₁₃ andGα_(q/11) activities and in hepatoma cells, AhR activity can agitatecell fate processes, whereby a persistent expression of AhR can promotetumor cell growth. The experiments were directed toward the molecularevents involved in Gα_(q/11) signaling.

In one embodiment modulation of AhR activity can inhibit or decreasecell growth. In another embodiment modulation of AhR activity can kill acell. In one embodiment modulation comprises down regulation of AhRprotein activity in a cell. In another embodiment modulation comprisesinhibition of AhR protein activity in a cell. In another embodimentmodulation comprises inhibition of AhR protein association with a Gprotein in a cell. In another embodiment modulation comprises downregulation of AhR gene expression in a cell. In one the cell is a tumorcell. In one embodiment the tumor is a lung, breast, colon, brain, boneliver, prostate, stomach, esophageal, skin or leukemia tumor cell. Inone embodiment the tumor is a solid tumor. In another embodiment thetumor is a liquid tumor. In one embodiment AhR activity is modulatedwith an AhR agonist. In another embodiment AhR activity is modulatedwith an AhR antagonist. In another embodiment AhR activity is modulatedwith a compound that has anti-estrogenic activity. In another embodimentAhR activity is modulated with a compound that has anti-androgenicactivity.

In one embodiment the tumor cell is in a mammal. In another embodimentthe tumor cell is in a human. In another embodiment a method fortreating a tumor in human is provided by administering a compound to thehuman that inhibits or decreases the activity of an AhR protein in thetumor. In another embodiment a method for treating a tumor in human isprovided by administering a compound to the human that inhibits ordecreases the gene expression of a AhR protein in the tumor.

For confocal immunofluorescence imaging microscopy, cells were treatedwith BBP for 5 and 15 min each followed by immunofluorescence stainingof both AhR and Gα_(q/11). In the absence of BBP, less expression ofboth AhR and Gα_(q/11) in the cytoplasm than in the nucleus was observed(FIG. 15a ). In cells stimulated by BBP, a clear increase in expressionof AhR in the nucleus and peri-nuclear regions at 5 min followed by anoutward spreading of AhR was observed at 15 min (FIG. 15b , firstcolumn). These results indicate a constitutive AhR activity andcytosolic translocation. With respect to expression of Gα_(q/11), itappeared to be stimulated in a similar way to that of AhR at 5 min (FIG.15b , second column). However, Gα_(q/11) had translocated from thecytosolic compartment towards the cell membrane at 15 min, supporting amaturation of GPCR-G protein complex capable of making a correcttransportation to the cell membrane based on the ontogenetic viewpoint,though the exact mechanism is unclear. Subsequently, siRNA knockout ofAhR suppressed the expression of nuclear AhR but not cytosolic AhR,which was confirmed by the knockout of AhR using scrambled siRNA (FIG.15c ). However, when BBP was added, AhR expression was increased in bothnucleus and peri-nuclear regions at 5 min, reaching a homeostatic stateby 15 min in the cytosol (FIG. 15d , first column). Notably, Gα_(q/11)was repressed by AhR siRNA (FIG. 15d , second column), which waspartially recovered by the addition of BBP at 5 min and totallyrecovered at 15 min, showing an apparent accumulation of Gα_(q/11) atthe cell membrane (FIG. 15d , second column). These results indicatedthat Gα_(q/11) is a downstream effector of memAhR. The dynamic movementsand constitutive activities of both AhR and Gα_(q/11) further suggest acompensatory effect, involving their activation, translocation andmaturation in the cell.

Because of the spatio-temporal dynamics, double immunogold transmissionelectron microscopy (IEM) was used to show interaction of memAhR at theplasma membrane. Cells were treated with BBP for 20 min and subjected toimmunocytochemistry using specific primary antibodies and secondaryantibodies of large gold particle-tagged Gα_(q/11) (20 nm in size) andsmall gold particle-tagged AhR (6 nm in size). The samples wereimmediately embedded in LR White Resin (Ted Pella, Redding, Calif.) andprepared for IEM. In the absence of ligand, three separateimmunogold-tagged Gα_(q/11) entities were displayed, including single,double and triple clusters at the cell membrane (FIG. 16a ), reflectingthe existence of different entities of GPCR-G protein complex. Treatingthe cells with BBP, a number of small gold-tagged AhR adhered to thelarge gold-tagged Gα_(q/11) was observed to form an AhR-Gα_(q/11)complex at the cell membrane (FIG. 16b ). In addition to the classicalmonomer and recently accepted dimers, the presence of polymericGPCR-Gα_(q/11) was observed at the cell membrane. This suggests avariety of conformational changes in GPCRs, including monomer, dimersand polymers (FIG. 16c ). The AhR-Gα_(q/11) complex was found mainly atthe plasma membrane. There were few in the cytosolic, but none in thenuclear compartment where abundant AhR and Gα_(q/11) existedindependently. No such AhR-Gα_(q/11) interaction was seen in the controlcells. The data revealed that the clusters of memAhR and GPCRs-Gα_(g/11)complex were not pre-coupled before ligand activation. Thepolymerization (either homo- or hetero-multimers) of GPCRs is meaningfulbecause it is an effective mode for modulating the function, subcellularlocalization, and biophysical properties of the interacting molecules.It probably enables the creation of more spatial docking sites for thescreening of exogenous ligands such as agonists and antagonists, orsynergistic bindings at the cell surface. Alternatively, it provides aclue into one of the most puzzling aspects of biological impact;specifically, how polycyclic aromatic hydrocarbon compounds in theenvironment are related to toxic, metabolic and carcinogenic responsesin cells.

To study the biochemical processes of G protein signaling, it wasverified that upon activation by BBP, memAhR can interact withGαq_(/11), as described previously. Subsequently, a decrease inphosphatidylinositol (PIP2) levels was observed resulting from thecleavage of PIP2 into two secondary messengers: diacylglycerol (DAG) andIP3 (FIG. 17a , first panel). IP3 is known to induce the release ofintracellular calcium through its receptor IP3R at the endoplasmicreticulum (FIG. 17a , second panel). Because G protein activation isoften accompanied by an influx of calcium ions, the origin of theBBP-elicited intracellular fluo-4-tagged Ca²⁺ levels was examined byreal-time live cell immunofluorescence imaging microscopy (FIG. 17b ,middle upper). The cells were cultivated in calcium free medium andfound the release of intracellular calcium (FIG. 17b , middle lower),indicating release from the internal calcium store. This result wasfurther confirmed by adding IP3R blocker 2-APB, which was found todose-dependently inhibit intracellular calcium levels (FIG. 17b , rightcolumn). An aberrant calcium release, however, can induce inflammatoryresponses⁴ and tumorigenesis. Accordingly, BBP was observed to induceproduction of COX-2 in 15 min, which could be blocked by adding 2-APB(FIG. 17c ), linking the increase in intracellular calcium with theactivation of COX-2. Moreover, BBP induced phosphorylation of anextracellular signal-regulated protein kinase ERK and activation ofCOX-2 (FIG. 17d ), which was blocked by chemical PD98059, a potent andselective noncompetitive inhibitor of the MAPK pathway (FIG. 17e ),indicating that ERK is the upstream activator of COX-2. To this end, BBPwas shown to induce the activation of COX-2 via the memAhR-activatedGα_(q/11) signaling in molecular processes. This indicates the existenceof a nongenomic AhR pathway because BBP significantly inhibited ARNTexpression, a gene encoding AhR nuclear translocator protein (FIG. 17f). This inhibitory effect can be interpreted as the action ofco-activated Gα₁₃ as described previously.

It is demonstrated that AhR can be a signal transducer in response toexternal signals, resulting in the excitation of GPCR-G proteinsignaling. It is proposed that the signal “pulls” the nearby cytosolicmemAhR (as activator) to the cell membrane to bind and activate thedissociated Gα_(q/11) (as effector), and “pushes” the downstreammolecular cascades for functions in human hepatic Huh-7 cancer cells.This “pull and push” model, as illustrated in FIG. 17g , contributesgreatly to the understanding of how the regulation of GPCR-G proteinsignaling is initiated and how the AhR-mediated signal transduction iscontrolled beyond the classical AhR pathway. The findings can furthermake an impact on the development of therapeutics focusing on themechanistic regulation of GPCRs and G proteins.

Cell Culture and Chemicals.

Huh-7 cells were obtained from the National Health Research Institute,Taiwan and cultured in DMEM (Gibco) supplemented with 10% fetal bovineserum (Gibco), 1% penicillin (100 U/mL), streptomycin (10 μg),amphotericin-α (0.25 mg) and grown at 37° C. in 5% CO₂. Culture mediaincluded BSS containing CaCl₂ (2 mM), D-glucose (5.5 mM), NaCl (130 mM),KCl (5.4 mM), HEPES (20 mM, pH 7.4) and MgSO₄ (1 mM). Calcium freemedium contained D-glucose (5.5 mM), NaCl (130 mM), KCl (5.4 mM), HEPES(20 mM, pH 7.4) and MgSO₄ (3 mM). Chemicals included were Fluo-4(Invitrogen), Benzyl butyl phthalate (BBP, Sigma), 2-aminoethoxydiphenylborate (2-APB, Sigma), ERK1/2 inhibitor: PD98059 (Calbiochem),6-diamidino-2-phenylindole (DAPI, Sigma). Antibodies included were AhR(Santa Cruz), Cox-2 (Minipore), Gα_(q/11) (sc-392) and Gβ (sc-378, SantaCruz), β-actin (Sigma), p44/42 MAPK (Erk1/2) (Cell Signaling),Phospho-α44/42 MAPK (Cell Signaling), Horseradish peroxidase(HRP)-labeled anti-mouse and anti-rabbit secondary antibodies (SantaCruz), Dye Light 488-conjugated secondary antibody (green color) and DyeLight 549-conjugated secondary antibody (red color) (Rockland).

hTS cells obtained from the preimplantation embryos in women with earlytubal ectopic pregnancy were described previously. Adherent hTS cellswere cultured in conditioned α-MEM containing 10 μg/ml bFGF (JRH,Biosciences, San Jose, Calif.), 10% FBS, and 1% penicillin-streptomycinat 37° C. in 5% CO₂. Cells were treated by RA (10 μM) for various timeintervals depending on the experiments.

RNA isolation and RT-PCR. Huh-7 cells (3×105) were seeded into a 6-welldish and incubated for 24 h. Cells cultured serum-free medium overnightwere treated with BBP (1 μM) for various time intervals. After BBPstimulation, cells were washed twice with PBS. Total RNAs were extractedby TRIzol methods (Invitrogen). RNA (2 μg) was used to synthesize cDNAby Reverse Transcription System (Promega). The c-DNAs were amplified bythe specific primers. The primer pairs were designed as follows: AhR,forward 5′-TAC TCT GCC GCC CAA ACT GG-3′ (SEQ ID NO: 49), reverse 5′-GCTCTG CAA CCT CCG ATT CC-3′ (SEQ ID NO: 50); β-actin, forward 5′-CTC GCTGTC CAC CTT CCA-3′ (SEQ ID NO: 51), reverse 5′-GCT GTC ACC TTC ACCGTTC-3′ (SEQ ID NO: 52). PCR conditions were set at 95° C. for 5 min and95° C. for 30 sec, 54° C. for 30 sec, 72° C. for 1 min followed by 72°C. for 10 min (36 cycles). The products were separated by 2% agarosegels and visualized by ethidium bromide.

Western Blotting Analysis.

Huh-7 cells (1×10⁶) were seeded into 10 cm dish and cultured overnight.The culture medium was changed to serum-free medium for another night.Cells were treated with BBP (1 μM) for various time intervals. For otherstudies, cells were pretreated with the chemical PD98059 (20 μM) or2-APB (30 μM) for 1 h followed by treatment with BBP. Cells were thenwashed twice with ice-cold PBS and lysed by RIPA lysis buffer(Minipore). Protein concentration was measured by BCA protein assay kit(Thermo). Equal amounts of protein (30 μg protein) were resolved by 8%SDS-PAGE, transferred onto PVDF membrane, and blocked with 5% non-fatdry milk for 1 h at room temperature. After blocking, the membrane wasincubated with the primary antibodies including AhR (1:1000), Cox-2(1:1000), Gα_(q/11) (1:100), Gβ (1:100), β-actin (1:5000), p44/42 MAPkinase (1:1000) or phospho-p44/42 MAP kinase (1:1000) overnight at 4° C.Cells were washed three times with PBST and then incubated with HRPconjugated secondary antibodies for 1 h at room temperature. Afterwashing, the blot was visualized using an enhanced chemiluminescence kit(ECL) (Amersham).

ChIP.

By using ChIP kit (Upstate Biotechnology, Lake Placid, N.Y.), cells wereserum-deprived for overnight and treated with RA (10 μM) for 4 hr. Forassay, briefly, the lysate was sonicated on ice to shear the DNA. Thecrosslinked chromatin was incubated with protein G agarose plus anti-RNApolymerase II (positive control), or normal mouse IgG (negative control)or primary antibody indicated. After sequential treatments with 5M NaCl,RNase A, EDTA, Tris, and proteinase K, the DNA mixtures were obtained byspin filter and subjected for polymerase chain reaction (PCR).

Immunoprecipitation.

Huh-7 cells were serum-deprived overnight and treated with BBP (1 μM)for 30 min. After pre-cleaning with protein G-agarose (Minipore) for 30min, specific antibody Gα_(q/11) or rabbit IgG was added to culturewhich was again incubated overnight. After incubation with proteinG-agarose for 2 h, the beads were washed three times with RIPA lysisbuffer, boiled in sample buffer, resolved by 8% SDS-PAGE and subjectedto AhR immunoblotting analysis.

Cells were serum-deprived overnight and treated with RA (10 μM) for 4hr. The cells were lysed by RIPA lysis buffer (Millipore). The mixturesof lysate and protein A or protein G agarose (Minipore) were incubatedwith rocking at 4° C. for 2 hr. Specific primary antibody or rabbit IgG(control) was added and incubated overnight. The immune protein complexwas then captured on beads with either protein A or protein G. Theantibody-bound proteins were precipitated by rocking for overnight. Theimmunoprecipitated proteins were washed with RIPA lysis buffer followedby analysis with SDS-PAGE and immunoblotting with another specificantibody to measure the interaction.

Immunofluorescence.

For immunocytochemistry, cells were fixed with 4% paraformaldehyde inPBS followed by permeabilization with 2% FBS/0.4% Triton X-100 in PBS(15 min). By 5% FBS blocking solution (2 hr) and rinsed three times,cells were incubated with specific primary antibody in PBS at 4° C.overnight. Appropriate FITC or PE or Texas Red conjugated secondaryantibody was added for 1 hr followed by DAPI staining for nucleus (5min) and subjected for microscopy.

Total Internal Reflection Fluorescence (TIRF) Microscopy.

Huh-7 cells were pre-transfected with pGFP-C1-AhR (a gift of H. Li) byusing LT1 transfection reagent (Minis Bio LLC, Madison, Wis.) for 24 h.For TIRF microscopy, cells were cultured in serum-free medium oncover-slip overnight followed by stimulation by BBP (1 μM, Sigma). Thedynamic activities of GFP-tagged AhR at the cell membrane were observedand analyzed by using Zeiss TIRF microscope with Axio Vision Rel. 4.8software.

Real-Time Live Cell Imaging Microscopy.

Cells were pre-treated with Fluo-4 (1 μM), a Ca²⁺-specific dye, in BSSbuffer at 37° C. for 20 min before treatment with BBP (1 μM).Measurements of relative intracellular calcium intensity were performedby real-time cell imaging microscopy and analyzed by Cell-R softwaresystem (Olympus). Either calcium-free medium or an IP3R inhibitor 2-APBused at various concentrations was used to test the intracellularcalcium responses in the cell culture.

Confocal Immunofluorescence Imaging Microscopy.

Cells with or without transfection by AhR siRNA were cultured andtreated with BBP (1 μM) for 5 and 15 min each. After treatment withprimary and secondary antibodies for AhR and Gα_(q/11), cells weresubjected to confocal immunofluorescence microscopy to analyze thedynamic movement in the cell compartments.

Double Immunogold Transmission Electron Microscopy.

Ultrathin sections of plastic embedded cells obtained by microwavefixation and processing²⁴ were pretreated with 5% sodium metaperiodate(10 min). The grids were incubated with an aliquot of IgG antibodyagainst AhR or Gα_(q/11) (C-19, sc-392, Santa Cruz) followed by probingwith a secondary anti-mouse IgG gold particles (6 nm in size) oranti-rabbit IgG gold particles (20 nm in size), respectively. Afterwashing, the sections were blocked by placing the grids on a drop of PBSwith 1% ovalbumin (15 min). Sections were then stained with uranylacetate and lead citrate and observed by transmission electronmicroscopy (Hitachi H-700 model, Japan).

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.Olanow, C. W. The scientific basis for the current treatment ofParkinson's disease. An. Rev. Med. 55, 41-60 (2004).

-   Freed, C. R. et al. Transplantation of embryonic dopamine neurons    for severe Parkinson's disease. N. Engl. J. Med. 344, 710-719    (2001).-   Lindvall, O. & Kokaia, Z. Stem cells for the treatment of    neurological disorders. Nature 441, 1094-1096 (2006).-   Kim, J. H. et al. Dopamine neurons derived from embryonic stem cells    function in an animal model of Parkinson's disease. Nature 418,    50-56 (2002).-   Bjorklund, L. M. et al. Embryonic stem cells develop into functional    dopaminergic neurons after transplantation in a Parkinson rat model.    Proc. Natl. Acad. Sci. USA 99, 2344-2349 (2002).-   Reubinoff, B. E., Itsykson, P., Turetsky T, Pera M F, Reinhartz E,    Itzik, A. & Ben-Hur, T. Neural progenitors from human embryonic stem    cells. Nat. Biotechnol. 19, 1134-1140 (2001).-   Roy, N. S., Cleren, C., Singh, S. K., Yang, L., Beal, M. F. &    Goldman, S. A. Functional engraftment of human ES cell-derived    dopaminergic neurons enriched by co culture with    telomerase-immortalized midbrain astrocytes. Nat. Med. 12, 1259-1268    (2006).-   Dunnett, S. B., Bjorklund A. & Lindvall, O. Cell therapy in    Parkinson's disease: stop or go? Nat. Rev. Neurosci. 2, 365-369    (2001).-   Parolini, O. et al. Concise review: Isolation and characterization    of cells from human term placenta: outcome of the first    international Workshop on Placenta Derived Stem Cells. Stem Cells    26, 300-311 (2008).-   Ilancheran. S. & Moodley, Y. & Manuelpillai, U. Human fetal    membranes: a source of stem cells for tissue regeneration and    repair? Placenta 30, 2-10 (2009).-   Surani, M. A., Hayashi, K. & Hajkova, P. Genetic and epigenetic    regulators of pluripotency. Cell 128, 747-762 (2007).-   Yamanaka, Y., Ralston, A., Stephenson, R. O., & Rossant, J. Cell and    molecular regulation of the mouse blastocyst. Dev. Dyn. 235,    2301-2314 (2006).-   Chen, H. F., Chao, K. H., Shew, J. Y., Yang, Y. S. & Ho, H. N.    Expression of leukemia inhibitory factor and its receptor is not    altered in the decidua and chorionic villi of human anembryonic    pregnancy. Hum. Reprod. 19, 1647-1654 (2004).-   Wånggren, K., Lalitkumar, P. G., Hambiliki, F., Ståbi, B.,    Gemzell-Danielsson, K. & Stavreus-Evers, A. Leukaemia inhibitory    factor receptor and gp130 in the human fallopian tube and    endometrium before and after mifepristone treatment and in the human    preimplantation embryo. Mol. Hum. Reprod. 13, 391-397 (2007).-   Keltz, M., Attar, E., Buradagunta, S., Olive, D., Kliman, H. &    Arici, A. Modulation of leukemia inhibitory factor gene expression    and protein biosynthesis in the human fallopian tube. Am. J. Obs.    Gyn. 175, 1611-1619 (1996).-   Smith, A. G., Heath, J. K., Donaldson, D. D., Wong, G. G., Moreau,    J., Stahl, M. & Rogers, D. Inhibition of pluripotential embryonic    stem cell differentiation by purified polypeptides. Nature 336,    688-690 (1998).-   Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A.,    Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D.,    Nicola, N. A. & Gough, N. M. Myeloid leukemia inhibitory factor    maintains the developmental potential of embryonic stem cells.    Nature 336, 684-687 (1988).-   Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S.,    Tweedie, S. & Smith, A. Functional expression cloning of Nanog, a    pluripotency sustaining factor in embryonic stem cells. Cell 113,    643-655 (2003).-   Boiani, L. A. & Scholer, H. R. Regulatory networks in embryo-derived    pluripotent stem cell. Nat. Rev. Mol. Cell. Biol. 6, 872-884 (2005).-   Adjaye, J. et al. Primary differentiation in the human blastocyst:    comparative molecular portraits of inner cell mass and trophectoderm    cells. Stem Cells 23, 1514-1525 (2005).-   He, S., Pant, D., Schiffmacher, A., Meece, A. & Keefer, C. L.    Lymphoid enhancer factor 1-mediated Wnt signaling promotes the    initiation of trophoblast lineage differentiation in mouse embryonic    stem cells. Stem Cells 26, 842-849 (2008).-   Maden, M. Retinoic acid in the development, regeneration and    maintenance of the nervous system. Nat. Rev. Neurosci. 8, 755-765    (2007).-   Wichterle, H., Lieberam, I., Porter, J. A. & Jessell, T. M. Directed    differentiation of embryonic stem cells into motor neurons. Cell.    110, 385-397 (2002).-   Li, X. J., Du, Z. W., Zarnowska, E. D., Pankratz, M., Hansen, L. O.,    Pearce, R. A. & Zhang, S. C. Specification of motorneurons from    human embryonic stem cells. Nat. Biotechnol. 23, 215-221 (2005).-   Zhang, X., Klueber, K. M., Guo, Z., Cai, J., Lu, C., Winstead, W.    I., Qiu, M. & Roisen, F. J. Induction of neuronal differentiation of    adult human olfactory neuroepithelial-derived progenitors. Brain    Res. 1073-1074, 109-119 (2006).-   Jacobs, S., Lie, D. C., DeCicco, K. L., Shi, Y., DeLuca, L. M.,    Gage, F. H. & Evans, R. M. Retinoic acid is required early during    adult neurogenesis in the dentate gyrus. Proc. Natl. Acad. Sci. USA.    103, 3902-3907 (2006).-   Tsai, Y.-L., Tseng, S.-F., Chang, S.-H., Lin, C.-C. & Teng, S.-C.    Involvement of replicative polymerases, Tel1p, Mec1p, Cdc13p, and    the Ku complex in telomere-telomere recombination. Mol. Cell. Biol.    22, 5679-5687 (2002).-   Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K.,    Yagi, R. & Rossant, J. Interaction between Oct3/4 and Cdx2    determines trophectoderm differentiation. Cell 123, 917-929 (2005).-   Cavaleri, F. & Scholer, H. R. (2003). Nanog: a new recruit to the    embryonic stem cell orchestra. Cell 113, 551-552 (2003).-   Martín-Ibáñez, R, Urbán, N., Sergent-Tanguy, S., Pineda, J. R.,    Garrido-Clua, N., Alberch, J. & Canals, J. M. Interplay of leukemia    inhibitory factor and retinoic acid on neural differentiation of    mouse embryonic stem cells. J. Neuron. Res. 85, 2686-2710 (2007).-   Bain, G., Kitchens, D., Yao, M., Huettner, J. E. & Gottlieb, D. I.    Embryonic stem cells express neuronal properties in vitro. Dev.    Biol. 168, 342-357 (1995).-   Tropepe, V., Hitoshi, S., Sirard, C., Mak, T. W., Rossant, J. & van    der Kooy, D. Direct neural fate specification from embryonic stem    cells: a primitive mammalian neural stem cell stage acquired through    a default mechanism. Neuron 30, 65-78 (2001).-   Smith, C. R., Chan, H. S. & deSa, D. J. Placental involvement in    congenital neuroblastoma. J. Clin. Pathol. 34, 785-789 (1981).-   Panicker, M. M. & Rao, M. Stem cells and neurogenesis. in Stem Cell    Biology (eds Msrshak, D. R., Gardner, R. L. & Gottlieb, D.) 399-438    (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,    2001).-   Yan, J., Tanaka, S., Oda, M., Makino, T., Ohgane, J. & Shiota, K.    Retinoic acid promotes differentiation of trophoblast stem cells to    a giant cell fate. Dev. Biol. 235, 422-432 (2001).-   Chen, L. & Khillan, J. S. Promotion of feeder-independent    self-renewal of embryonic stem cells by retinol (vitamin A). Stem    Cells 26, 1858-1864 (2008).-   Li, L. et al. Human Embryonic Stem Cells Possess Immune-Privileged    Properties. Stem Cells 22, 448-456 (2004).-   Swijnenburg, R. J. et al. Immunosuppresive therapy mitigates    immunological rejection of human embryonic stem cell xenografts.    Proc. Natl. Acad. Sci. USA. 105, 12991-12996 (2008).-   Bavaresco, L., Bernardi, A., Braganhol, E., Cappellari, A. R.,    Rockenbach, L., Farias, P. F., Wink, M. R., Delgado-Cañedo, A. &    Battastini, A. M. The role of ecto-5′-nucleotidase/CD73 in glioma    cell line proliferation. Mol. Cell. Biochem. 319, 61-68 (2008).-   Napoli, I. & Neumann, H. Microglial clearance function in health and    disease. Neuroscience 158, 1030-1038 (2009).-   Song, H., Stevens, C. F. & Gage, F. H. Astroglia induce neurogenesis    from adult neural stem cells. Nature 417, 39-44 (2002).-   Annerén, C., Cowan, C. A & Melton, D. A. The Src family of tyrosine    kinases is important for embryonic stem cell self-renewal. J. Biol.    Chem. 279, 590-598 (2004).-   Torres, J. & Watt, F. M. Nanog maintains pluripotency of mouse    embryonic stem cells by inhibiting NFkappaB and cooperating with    Stat3. Nat. Cell Biol. 10, 194-201 (2008).-   Myers, R., L., Ray, S. K., Eldridge, R., Chotani, M. A., Chiu, I-M.    Functional characterization of the brain-specific FGF-1 promoter,    FGF-1B. J. Biol. Chem. 270, 8257-8266 (1995).-   Wu, R. M., Murphy, D. L. & Chiueh, C. C. Suppression of hydroxyl    radical formation and protection of nigral neurons by 1-deprenyl    (Selegiline). Ann. N.Y. Acad. Sci. 786, 379-389 (1996).-   Götz, M. Glial cells generate neurons—master control within CNS    regions: developmental perspectives on neural stem cells.    Neuroscientist 9, 379-97 (2003).-   Singh, S. K., Hawkins, C., Clarke, I. D., Squire, J. A., Bayani, J.,    Hide, T., Henkelman, R. M., Cusimano, M. D. & Dirks, P. B.    Identification of human brain tumour initiating cells. Nature 432,    396-401 (2004).-   Zhu, Q. F., Ma, J., Yu, L. I. & Yuan, C. G. Grafted neural stem    cells migrate to substantia nigra and improve behavior in    Parkinsonian rats. Neurosci. Lett. 462, 213-218 (2009).-   Lindvall O, Kokaia Z. & Martinez-Serrano A. Stem cell therapy for    human neurodegenerative disorders-how to make it work. Nat. Med. 10    (Suppl), S42-50 (2004).-   Wagner, J. et al. Induction of a midbrain dopaminergic phenotype in    Nurr1-overexpressing neural stem cells by type 1 astrocytes. Nat.    Biotechnol. 17, 653-659 (1999).

While some embodiments have been shown and described herein, it will beobvious to those skilled in the art that such embodiments are providedby way of example only. Numerous variations, changes, and substitutionswill now occur to those skilled in the art without departing from theinvention. It should be understood that various alternatives to theembodiments of the invention described herein can be employed inpracticing the invention. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. An isolated human neural stem cell produced froman isolated human trophoblastic stem cell, wherein the isolated humanneural stem cell expresses Cdx2, Nanog, Ngn3, RARβ, RXRα, RXRβ, RALDH-2,and RALDH-3 proteins, and wherein the isolated human neural stem cellhas low levels of expression or an absence of expression of CD33 orCD133 cell surface proteins as compared to the isolated humantrophoblastic stem cell.